Expression of Cry3B insecticidal protein in plants

ABSTRACT

The present invention discloses methods and compositions comprising a group of novel expression cassettes which provide significantly improved levels of accumulation of Coleopteran inhibitory Cry3B and Cry3B variant amino acid sequences when these are expressed in plants. The preferred embodiments of the invention provide at least up to ten fold higher levels of insect controlling protein relative to the highest levels obtained using prior compositions. In particular, transgenic maize expressing higher levels of a protein designed to exhibit increased toxicity toward Coleopteran pests deliver superior levels of insect protection and are less likely to sponsor development of populations of target insects that are resistant to the insecticidally active protein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention discloses transgenic plants expressingsubstantially higher levels of insect controlling Bacillus thuringiensisδ-endotoxin. Methods for obtaining such plants and compositions, andmethods for using such plants and compositions are described. Alsodisclosed are improved polynucleotide cassettes containing preferredprotein coding sequences which impart the substantially higher levels ofinsect controlling δ-endotoxins. The preferred embodiments of theinvention surprisingly provide up to ten fold higher levels of insectcontrolling protein relative to the highest levels obtained using priorcompositions. In particular, transgenic maize expressing higher levelsof a protein designed to exhibit increased toxicity toward Coleopteranpests deliver superior levels of insect protection and are less likelyto sponsor development of populations of target insects that areresistant to the insecticidally active protein.

2. Description of the Related Art

Almost all field crops, plants, and commercial farming areas aresusceptible to attack by one or more insect pests. Particularlyproblematic are Coleopteran and Lepidopteran pests. Because crops ofcommercial interest are often the target of insect attack,environmentally-sensitive methods for controlling or eradicating insectinfestation are desirable. This is particularly true for farmers,nurserymen, growers, and commercial and residential areas which seek tocontrol insect populations using ecologically friendly compositions.

The most widely used environmentally-sensitive insecticidal formulationsdeveloped in recent years have been composed of microbial proteinpesticides derived from the bacterium Bacillus thuringiensis, aGram-positive bacterium that produces crystal proteins or inclusionbodies which are specifically toxic to certain orders and species ofinsects. Many different strains of B. thuringiensis have been identifiedwhich produce one or more insecticidal crystal proteins as well as otherinsecticidal non-crystal forming proteins. Compositions including B.thuringiensis strains which produce insecticidal proteins have beencommercially available and used as environmentally acceptableinsecticides because they are quite toxic to specific target insectpests, but are harmless to plants and to vertebrate and invertebrateanimals. More importantly, because these insect controlling proteinshave to be ingested by susceptible target insect pests in order to exerttheir insecticidal or toxic effects, judicious application of suchprotein compositions limits or prevents non-target insect members of thesusceptible order which may also be susceptible to the composition fromsignificant exposure to the proteins (for example, non-targetLepidopteran species where Lepidopteran specific B.t. crystal protein isused in an insecticidal formulation). Additionally, insects of variousorders have been shown to totally lack susceptibility to specificallytargeted insecticidal proteins even when ingested in large amounts.

δ-ENDOTOXINS

δ-endotoxins are used to control a wide range of plant-eatingcaterpillars and beetles, as well as mosquitoes. These proteins, alsoreferred to as insecticidal crystal proteins, crystal proteins, and Bttoxins, represent a large collection of insecticidal proteins producedby B. thuringiensis that are toxic upon ingestion by a susceptibleinsect host. Over the past decade research on the structure and functionof B. thuringiensis toxins has covered all of the major toxincategories, and while these toxins differ in specific structure andfunction, general similarities in the structure and function areassumed. A recent review describes the genetics, biochemistry, andmolecular biology of Bt toxins (Schnepf et al., Bacillus thuringiensisand its Pesticidal Crystal Proteins, Microbiol. Mol. Biol. Rev.62:775-806, 1998). Based on the accumulated knowledge of B.thuringiensis toxins, a generalized mode of action for B. thuringiensistoxins has been created and includes: ingestion by the insect,solubilization in the insect midgut (a combination stomach and smallintestine), resistance to digestive enzymes sometimes with partialdigestion by gut specific proteases catalyzing specifically a cleavageat a peptide site within a protoxin structure which “activates” thetoxin, binding of the toxin to the midgut cells' brush border, formationof a pore in the insect midgut cell, and the disruption of cellularhomeostasis (English and Slatin, 1992).

GENES ENCODING CRYSTAL PROTEINS

Many of the δ-endotoxins are related to various degrees by similaritiesin their amino acid sequences. Historically, the proteins and the geneswhich encode them were classified based largely upon their spectrum ofinsecticidal activity. A review by Höfte and Whiteley (1989) discussesthe genes and proteins that were identified in B. thuringiensis prior to1990, and sets forth the nomenclature and classification scheme whichhas traditionally been applied to B. thuringiensis genes and proteins.The original nomenclature took advantage of the discovery that the fewBt Cry proteins known at the time generally fell into a limited numberof classes, wherein each class represented proteins having specificityfor specific orders of insects. For example, cry1 genes encodedLepidopteran-toxic Cry1 proteins. cry2 genes encoded Cry2 proteins thatwere generally toxic to both Lepidopterans as well as to Dipterans. cry3genes encoded Coleopteran-toxic Cry3 proteins, while cry4 genes encodedDipteran-specific toxic Cry4 proteins. The nomenclature has, for thepast decade or more become rather confusing with the discovery of moredistantly related classes of insecticidal Bt proteins. More recently, asimplified homogeneous nomenclature and basis for classifications of Btproteins has been adopted and has been reviewed by Schnepf et al.(1998). Schnepf et al. (1998) also provides a structural solution for aCry1 crystal. This simplified nomenclature will be adopted herein. Theconvention of identifying Bt genes with lower case, italicized letters(eg. cry1Ab1) and identifying Bt proteins with uppercase first character(eg. Cry1Ab1) will also be observed herein.

Based on the degree of sequence similarity, the proteins have beenfurther classified into subfamilies. Proteins which appeared to be moreclosely related within each family were assigned divisional letters suchas Cry1A, Cry1B, Cry1C, etc. Even more closely related proteins withineach division were given names such as Cry1Ca, Cry1Cb, etc. and stilleven more closely related proteins within each division were designatedwith names such as Cry1Bb1, Cry1Bb2, etc.

The modern nomenclature systematically classifies the Cry proteins basedupon amino acid sequence homology rather than upon insect targetspecificities. The classification scheme for many known toxins, notincluding allelic variations in individual proteins, is summarized inregularly updated tables which can be obtained from Dr. Neil Crickmoreat at the biology department of Sussex University in Great Britain.

BIO-INSECTICIDE POLYPEPTIDE COMPOSITIONS

The utility of bacterial crystal proteins as insecticides was extendedbeyond Lepidopterans and Dipteran larvae when the first isolation of aColeopteran-toxic B. thuringiensis strain was reported (Krieg et al.,1983; 1984). This strain (described in U.S. Pat. No. 4,766,203,specifically incorporated herein by reference), designated B.thuringiensis var. tenebrionis, was reported to be toxic to larvae ofthe Coleopteran insects Agelastica alni (blue alder leaf beetle) andLeptinotarsa decemlineata (Colorado potato beetle).

U.S. Pat. No. 5,024, 837 also describes hybrid B. thuringiensis var.kurstaki strains which showed activity against Lepidopteran insects.U.S. Pat. No. 4,797,279 (corresponding to EP 0221024) discloses a hybridB. thuringiensis containing a plasmid from B. thuringiensis var.kurstaki encoding a Lepidopteran-toxic crystal protein-encoding gene anda plasmid from B. thuringiensis tenebrionis encoding a Coleopteran-toxiccrystal protein-encoding gene. The hybrid B. thuringiensis strainproduces crystal proteins characteristic of those made by both B.thuringiensis kurstaki and B. thuringiensis tenebrionis. U.S. Pat. No.4,910,016 (corresponding to EP 0303379) discloses a B. thuringiensisisolate identified as B. thuringiensis MT 104 which has insecticidalactivity against Coleopterans and Lepidopterans. More recently, Osman etal. disclosed a natural Bacillus thuringiensis isolate which displayedactivity against at least two orders of insects and against nematodes(WO 98/30700).

It has been known for more than two decades that compositions comprisingBt insecticidal proteins are effective in providing protection frominsect infestation to plants treated with such compositions. Morerecently, molecular genetic techniques have enabled the expression of Btinsecticidal proteins from nucleotide sequences stably inserted intoplant genomes (Perlak et al., Brown & Santino, etc.). However,expression of transgenes in plants has provided an avenue for increasedinsect resistance to Bt's produced in plants because plants have notbeen shown to produce high levels of insecticidal proteins. It wasinitially believed that gross morphological or topological differencesin gene structure and architecture between plant and bacterial systemswas the limitation which prevented over-expression of Bt transgenes inplants. These differences were seemingly overcome as disclosed by Perlaket al. (U.S. Pat. No. 5,500,365) and by Brown et al. (U.S. Pat. Nos.5,424,412 and 5,689,052) wherein transgenes encoding Bt insecticidalprotein which contained plant preferred codons were shown to improve thelevels of expression. Alternatively, truncating the protoxin codingdomain to the shortest peptide coding domain which still encoded aninsecticidal protein was also deemed sufficient to overcome thelimitation of vanishingly low expression levels of the Bt encodingtransgene in planta. Expression levels of Bt proteins in planta fromtransgenes has varied widely independent of the means used forexpression, and accumulated protein levels have ranged from virtuallyundetectable to 2 parts per million to around 20 to 30 parts permillion. However, even though all of these approaches provided improvedlevels of Bt protein accumulation in plants, none provided levels ofexpression which could ensure that insect resistance would not become aproblem without the necessity of coordinate expression of one or moreadditional insecticidal toxins by the transgenic plant, or alternativelywithout the coordinate topical application of additional supplemental Btor insecticidal chemical compositions.

The importance of accumulation of higher levels of Bt toxin forpreventing insect resistance to individual Bt toxins has been understoodfor some time. Various laboratory studies in which selection against Btwas applied over several generations of insects have confirmed thatresistance against Bt insecticidal proteins is seldom obtained. Itshould be emphasized that laboratory conditions represent rather low butconstant selection pressure conditions, allowing for the survival of asub-population of insects which have been subjected to insecticidalpressure and which produce the subsequent generations of insects.Succeeding generations are also maintained on media containing low butconstant concentrations of insecticidal protein. Generally,concentrations used for selection pressures range from LC40 to aroundLC60 or so, however, LC95 concentrations have also tested for thedevelopment of resistance. In most cases, resistance is acquired slowly,generally developing within a reasonably few generations, for example10-50 generations. However, such resistance is not observed wheresubstantially higher levels of toxin are used, or in situations in whichmultiple toxins are provided.

At present, recombinant plants expressing commercially useful levels ofBt insecticidal protein generally contain only one gene encoding asingle class of Bt. Such plants are anticipated to have a very limitedduration of use for two reasons. First, these plants are expressinginsufficient levels of the insecticidal protein to ensure that alltarget insects exposed to and feeding from the plant tissues willsuccumb due to the dose of toxin ingested. Second, because of theinsufficient insecticidal protein levels, the potential for developmentof resistance is unreasonably increased. This is not to say that thelevel of toxin produced by such transgenic plants is insufficient to beeffective. This merely represents the limitations of expression ofδ-endotoxins in planta even when using sequences encoding Bt δ-endotoxinwhich have been modified to conform to plant preferred sequences. Onelimitation which has been observed for many Bt δ-endotoxin encodingsequences modified for expression in plants is that is has beenimpossible to predict which Bt δ-endotoxin would be effective forexpression in plants. (For example, expression of Cry2Aa in cottonplants results in phytotoxicity when targeted to the chloroplast,however expression of a closely related cry2Ab sequence is notphytotoxic when targeted to the chloroplast. (Corbin et al., U.S. patentapplication Ser. No. 09/186,002 ). Even so, levels of δ-endotoxinprotein produced in plants is not sufficient to be effective against alldesired target insect species known to be susceptible to a given typeand class of δ-endotoxin.

As indicated above, alternative approaches to development of resistanceto insecticidal proteins has included ineffective attempts to increasethe expression levels of transgenes in plants. Alternatively, additionalinsecticidal genes could be engineered into plants so that multipletoxins are coordinately expressed. This would provide a more effectivemeans for delaying the onset of resistance to any one combination ofBt's, however, this still does not overcome the limitation ofinsufficient levels of insecticidal protein accumulating in therecombinant plant(s). An additional alternative to insufficient levelsof expression has been to engineer genes encoding Bt insecticidalcrystal proteins which demonstrate improved insecticidal properties,having either a broader host range or an increased biological activity,which could conceivably result in requiring less of the recombinantprotein to control a target insect species than was required of thenative form of the protein.

The combination of structural analyses of B. thuringiensis toxinsfollowed by an investigation of the function of such structures, motifs,and the like has taught that specific regions of crystal proteinendotoxins are, in a general way, responsible for particular functions.

Domain 1, for example, from Cry3Bb and Cry1Ac has been found to beresponsible for ion channel activity, the initial step in formation of apore (Walters et al., 1993; Von Tersch et al., 1994). Domains 2 and 3have been found to be responsible for receptor binding and insecticidalspecificity (Aronson et al., 1995; Caramori et al., 1991; Chen et al.1993; de Maagd et al., 1996; Ge et al., 1991; Lee et al., 1992; Lee etal., 1995; Lu et al., 1994; Smedley and Ellar, 1996; Smith and Ellar,1994; Rajamohan et al., 1995; Rajamohan et al., 1996; Wu and Dean,1996). Regions in domain 2 and 3 can also impact the ion channelactivity of some toxins (Chen et al., 1993, Wolfersberger et al., 1996;Von Tersch et al., 1994).

Unfortunately, while many investigators have attempted, few havesucceeded in making mutated crystal proteins with improved insecticidaltoxicity. In almost all of the examples of genetically-engineered B.thuringiensis toxins in the literature, the biological activity of themutated crystal protein is no better than that of the wild-type protein,and in many cases, the activity is decreased or destroyed altogether(Almond and Dean, 1993; Aronson et al., 1995; Chen et al., 1993, Chen etal., 1995; Ge et al., 1991; Kwak et al., 1995; Lu et al., 1994;Rajamohan et al., 1995; Rajamohan et al., 1996; Smedley and Ellar, 1996;Smith and Ellar, 1994; Wolfersberger et al., 1996; Wu and Aronson,1992). However, Van Rie et al. have recently accomplished theimprovement of a Cry3A δ-endotoxin having increased Coleopteraninsecticidal activity by identifying a single mutant having increasedinsecticidal activity. Van Rie et al. propose a method for identifyingmutants having increased insecticidal activity in which the methodconsists of identifying amino acid mutations which decrease theinsecticidal activity, and selectively altering those residues by sitedirected mutagenesis to incorporate one or more of the naturallyoccurring 20 amino acids at those positions, and feeding the variousforms of the resulting altered protein to western or northern cornrootworms to identify those having improved activity (U.S. Pat. No.5,659,123). While no sequences were enabled using the method, asmentioned above, Van Rie et al. succeeded in identifying only onesequence having increased activity and did not demonstrate an increasein expression of the mutant form as compared to the native sequence.

For a crystal protein having approximately 650 amino acids in thesequence of its active toxin, and the possibility of 20 different aminoacids at each position in this sequence, the likelihood of arbitrarilycreating a successful new structure is remote, even if a generalfunction to a stretch of 250-300 amino acids can be assigned. Indeed,the above prior art with respect to crystal protein gene mutagenesis hasbeen concerned primarily with studying the structure and function of thecrystal proteins, using mutagenesis to perturb some step in the mode ofaction, rather than with engineering improved toxins.

Collectively, the limited successes in the art to develop non-naturallyoccurring toxins with improved insecticidal activity have stifledprogress in this area and confounded the search for improved endotoxinsor crystal proteins. Rather than following simple and predictable rules,the successful engineering of an improved crystal protein may involvedifferent strategies, depending on the crystal protein being improvedand the insect pests being targeted. Thus, the process is highlyempirical.

Accordingly, traditional recombinant DNA technology is clearly notroutine experimentation for providing improved insecticidal crystalproteins. What has been lacking in the prior art are rational methodsfor producing genetically-engineered B. thuringiensis crystal proteinsthat have improved insecticidal activity and, in particular, improvedtoxicity towards a wide range of Lepidopteran, Coleopteran, or Dipteraninsect pests. Methods and compositions which address these concerns weredisclosed in U.S. Pat. No. 6,063,597 (filed Dec. 18, 1997; English etal.) and other related U.S. Pat. No. 6,060,594, filed Dec. 18, 1997,English et al.; U.S. Pat. No. 6,077,824, filed Dec. 18, 1997, English etal.; and U.S. Pat. No. 6,023,013, filed Dec. 18, 1997, English et al.)and in Van Rie et al. (U.S. Pat. No. 5,659,123, Jun. 1, 1999). Inaddition, recombinantly improved δ-endotoxins have continued to beexpressed poorly and/or cause phytoxic effects when expressed in plants,thus leading to the recovery of fewer commercially useful transgenicevents.

SUMMARY OF THE INVENTION

Described herein are novel compositions and methods for expressing intransformed plants variant Cry3 B. thuringiensis δ-endotoxins havingsignificant Coleopteran inhibitory activity. These compositions andmethods advantageously result in plants expressing B. thuringiensis Cry367-endotoxins at increased levels not previously observed for Cryδ-endotoxins. Increased levels of Cry3 δ-endotoxin expression arereflected in the attainment of higher maximal expression levels inindividual transgenic insertion events. Unexpectedly, the particularcompositions disclosed herein result in the recovery of an increasedpercentage of transgenic events which manifest expression levels thatfar exceed threshold levels of expression necessary for Coleopteraninsect control and which provide sufficient toxin levels capable ofsupporting a resistance management strategy. Since Cry3 δ-endotoxins aretypically less potent than other δ-endotoxins commonly used to controlLepidopteran or Dipteran target pests when expressed in transgenicplants, attainment of higher maximal levels of Cry3 δ-endotoxinexpression and recovery of more transgenic events with effectiveexpression levels are both critical in isolating transgenic eventsexpressing Cry3 δ-endotoxin which exhibit commercially useful levels oftarget insect control.

Another limitation of the prior art addressed by the present inventionis the development of insect resistance to δ-endotoxins provided byplant expression. Specifically, the instant invention provides asuperior strategy for the delay or elimination of the development ofresistance to Cry3 δ-endotoxins through improved accumulation ofδ-endotoxin within plant cells so that levels of the δ-endotoxin aremaintained in-planta above a threshold level of protein, typicallymeasured in parts per million (ppm). Improved expression ofδ-endotoxins, which also should be taken to mean increased expression inview of what has been previously observed in the art, is believed toresult in delayed onset of insect resistance and thus extends theutility of plant expressed δ-endotoxins as insect control agents.

In preferred embodiments, the present invention provides isolated andpurified novel Cry3B δ-endotoxin proteins exhibiting particularlyeffective insecticidal activity directed toward controlling Coleopteranpest insect species. Such δ-endotoxin proteins of the present inventionare provided by expression from isolated, purified and improved orenhanced DNA or polynucleotide sequences each comprising a Cry3δ-endotoxin coding sequence placed under the control of preferred plantfunctional gene expression elements such as a promoter, an untranslatedleader sequence, an intron and a transcription termination andpolyadenylation sequence. Some preferred DNA or polynucleotide sequencesmay also provide for plastid or chloroplast targeting protein sequences.Preferred DNA constructs of the present invention include thoseconstructs which encode Cry3 δ-endotoxins exhibitingColeopteran-inhibitory or Coleopteran-controlling activity. In anillustrative embodiment, polynucleotide sequences are assembled into anexpression cassette for introduction into plant genomic DNA, wherein theexpression cassette comprises a Cry3Bb δ-endotoxin variant codingsequence operably linked to a sequence comprising a promoter, anuntranslated leader sequence, an intron and a transcription terminationand polyadenylation sequence. In particular, a transgene localizedwithin a plant operable polynucleotide expression cassette orpolynucleotide sequence comprising an expression cassette which iscomprised of genetic elements which function in plant cells to express adesired protein from a nucleic acid coding sequence (the transgene)which is operably localized within said expression cassette. The codingsequence is linked upstream to at least a promoter sequence, anuntranslated leader sequence (UTL), an intron sequence, and in-frame incertain indicated embodiments to a sequence encoding a plastid orchloroplast targeting peptide. The coding sequence is also linkeddownstream to at least a plant functional transcription termination andpolyadenylation sequence. Polynucleotide sequences comprising such anexpression cassette are shown herein to improve expression of thedesired protein encoded from within the cassette, improve the number ofevents obtained from the use of the polynucleotide sequence in planttransformation, wherein said improved number of events contain thedesired transgene localized within the expression cassette and exhibitimproved levels of expression of one or more desired proteins. Theimproved number of events are also surprisingly observed to express thedesired protein at levels above 2 to 5 parts per million but in generalbelow 200 to 500 parts per million of total cell protein. Even moresurprising were some events in particular which expressed the desiredprotein at levels well above 500 ppm. Indicated embodiments disclose asequence encoding a variant Cry3Bb δ-endotoxin comprising the isolatedand purified SEQ ID NO:9, from NcoI to EcoRI as set forth in FIG. 1illustrating plasmid pMON25096. Yet other embodiments disclose a variantCry3Bb δ-endotoxin coding sequence comprising an isolated and purifiedSEQ ID NO:11, from NcoI to EcoRI as set forth in FIG. 2 illustratingplasmid pMON33741. It is contemplated, however, that any Cry3δ-endotoxin exhibiting substantial Coleopteran-inhibitory orColeopteran-controlling activity greater than or equal to that disclosedin the present invention could be utilized according to the embodimentsof the present invention, with those Cry3 proteins bearing substantialhomologies to Cry3Bb being particularly preferred.

In a preferred embodiment, the invention provides for transgenic plantswhich have been transformed with a DNA construct or expression cassetteof the present invention that is expressed and translated atunexpectedly high levels by the plant which results in surprisingly highlevels of δ-endotoxin accumulation. Monocotyledenous plants may betransformed according to the methods and with the DNA constructsdisclosed herein. However, it is also anticipated that dicotyledenousplants could also be transformed with DNA sequences disclosed herein byone skilled in the art in order to obtain transgenic plants providingunexpectedly useful levels of insect resistance without the risk ofdevelopment of insect resistance to the δ-endotoxin. The planttransformed by the instant invention may be prepared, in a furtherpreferred embodiment, by a process including obtainment of the isolatedand purified DNA construct contained within the expression cassette, andthen transforming the plant with the construct so that the plantexpresses the protein for which the construct encodes. Alternatively,the plant transformed by the instant invention may be prepared, in afurther preferred embodiment, by a process including introduction of theisolated and purified DNA construct into a transformation competentAgrobacterium strain, and then transforming the plant with theAgrobacterium strain containing the construct so that the plantexpresses the proteins for which the construct encodes. It has beenobserved herein that transformation of plants by the disclosedcompositions and methods results surprisingly in increased frequenciesof transformants exhibiting transgene expression as well as in therecovery of individual transgenic events exhibiting unexpectedly higherabsolute levels of transgene expression.

It is contemplated that the increased expression levels observed in thedisclosed invention will allow for reduced development of insectresistance to Bt δ-endotoxins presented to target insect pests. This maybe achieved by transforming a plant with the preferred DNA construct toachieve high rates of Cry3 expression alone, or by simultaneouslyexposing target insects to the disclosed Cry3 δ-endotoxins along withother compositions effective in controlling Coleopteran species such asvariants of Cry3B (English et al., WO 99/31248), variant Cry3A orvariant Cry3D (U.S. Pat. No. 5,659,123), CryET33 and CryET34 (Donovan etal., WO 97/17600), CryET70 (U.S. application Ser. No. 09/184,748; Mettuset al., Nov. 2, 1998), Cry6A, Cry6B, Cry8B (U.S. Pat. No. 5,277,905),CryET29 (Rupar et al., WO 97/21587), insecticidal acyl lipid hydrolases,combinations of amino acid oxidases and tedanalactam synthases (Romanoet al., U.S. application Ser. No. 09/063,733, filed Apr. 21, 1998), orinsecticidal proteins such as VIP1 (Gay, WO 97/26339; Gourlet et al., WO98/02453) and VIP3 (Estruch et al., U.S. Pat. No. 5,877,012; 1999) amongothers. Susceptible target insects include Diabroticus spp. Wire Worm inZea mays and Leptinotarsa decemlineata (Say) in Solanum tuberosum, andBoll Weevil in Gossypium species (cotton).

It is therefore contemplated that the compositions and methods disclosedby the present invention will provide many advantages over the prior artincluding those specifically outlined above. Other advantages includeimproved control of susceptible target insect pests and achieving seasonlong protection from insect pathogens. An additional advantage of thepresent invention provides for reducing the number of transgenic eventsthat have to be screened in order to identify one which containsbeneficial levels of one or more insect controlling compositions. Thepresent invention also encompasses cells transformed with the DNAconstructs disclosed herein. Also, transformation vectors such asplasmids, bacmids, artificial chromosomes, viral vectors and such arecontemplated as elements for use in delivering the nucleotidecompositions of the present invention into contemplated cells in orderto obtain transformed host cells, both prokaryotic and eukaryotic, whichexpress the δ-endotoxin proteins encoded by the novel DNA constructdisclosed herein. It is further contemplated that in some instances thegenome of a transgenic plant of the present invention will have beenaugmented through the stable integration of an expression cassetteencoding a Coleopteran inhibitory or controlling B. thuringiensisδ-endotoxin or variants thereof as described herein. Furthermore, morethan one transgene encoding an insecticidal composition will beincorporated into the nuclear genome, or alternatively, into thechloroplast or plastid genome of the transformed host plant cell. It isenvisioned that more than one polynucleotide encoding an insecticidalcrystal protein will be incorporated into the genome of a plant cell andit may be desirable to have two or even more sequences encodinginsecticidal or other plant beneficial proteins within the nucleotidesequences contained within the cell. Such recombinantly derived proteinsmay exist as precursors, pro-toxins, or as fusions of beneficialproteins linked by flexible amino acid linker sequences or by proteasespecific cleavage sequences well known in the art. Chimeras comprisingfusions of insecticidal proteins are also envisioned. The offspring oftransgenic plant host cells can be manipulated artificially to producewhole recombinant plants exhibiting improved insecticidal properties,and the recombinant nucleotide sequences are shown herein to beheritable. The heritability of the elements is a preferred aspect ofthis invention, so that the expression elements are able to be deliveredto lineal descendants of the original transformed host plant cell,giving rise first to a stably transformed plant whose constituent cellsexpress the desired transgene, albeit tissue specific expression can beselectively manipulated generally through the choice of plant operablepromoter selected for use in a given expression cassette, as describedabove. Transformed plants give rise to seeds containing the heritableexpression cassette, and the seeds thus give rise to plants in linealfashion which contain the expression cassette, generally in Mendelianfashion, particularly when selfed according to well known methods in theart.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates plasmid pMON25096.

FIG. 2 illustrates plasmid pMON33741.

FIG. 3 illustrates plasmid pMON25097.

FIG. 4 illustrates plasmid pMON33748.

FIGS. 5A-5F illustrates the nucleotide and amino acid sequencetranslation of a variant Cry3Bb.11098 insecticidal protein as shown inSEQID NO:9.

FIGS. 6A-6F illustrates the nucleotide and amino acid sequencetranslation of a variant Cry3Bb.11231 insecticidal protein as shown inSEQID NO:11.

DETAILED DESCRIPTION OF THE INVENTION

The following detailed description of the invention is provided to aidthose skilled in the art in practicing the present invention. Even so,the following detailed description should not be construed to undulylimit the present invention as modifications and variations in theembodiments discussed herein may be made by those of ordinary skill inthe art without departing from the spirit and scope of the presentinvention.

DEFINITIONS

The following words and phrases have the meanings set forth below.

Biological functional equivalents. As used herein such equivalents withrespect to the insecticidal proteins of the present invention arepeptides, polypeptides and proteins that contain a sequence or moietyexhibiting sequence similarity to the novel peptides of the presentinvention, such as Cry3Bb.11231, and which exhibit the same or similarfunctional properties as that of the polypeptides disclosed herein,including insecticidal activity. Biological equivalents also includepeptides, polypeptides and proteins that react with, i.e. specificallybind to antibodies raised against Cry3Bb and that exhibit the same orsimilar insecticidal activity, including both monoclonal and polyclonalantibodies.

Combating or Controlling Insect Damage in an agricultural context refersto reduction of damage in relative units to a crop or plant part causedby infestation of an insect pest. More generally, this phrase refers toreduction in the adverse effects caused by the presence of an undesiredinsect in any particular location.

Event refers to a transgenic plant derived from one of the following:

1. the insertion of foreign DNA into one or more unique sites in thenuclear genomic DNA;

2. the insertion of foreign DNA into one or more unique sites in theplastid, chloroplast or mitochondrial genome;

3. the introduction of a stable, heritable, epigenetic vector into thecytoplasm of a plastid, chloroplast, or mitochondria; or

4. a combination of any of the foregoing processes.

Events derived from these processes contain an expression cassetteexpressing a desired coding sequence as described herein. Events arealso referred to as ITE's (independent transformation events).

Expression: The combination of intracellular processes, includingtranscription, translation, and other intracellular protein and RNAprocessing and stabilization functions, undergone by a nucleic acidcoding sequence controlled by genetic sequences which function in plantcells to achieve production of a desired product, such as a structuralgene encoding an RNA molecule, or an RNA molecule being used as asubstrate for a reverse transcriptase enzyme or enzyme complex.

Improved or enhanced expression cassette refers to the specificcombination and order of genetic elements associated with theinsecticidal protein encoding sequence which, when expressed within aplant cell:

gives rise to the surprising average level of that protein expressed inplants, plant tissue, or plant cells;

gives rise to the unexpected number of transformation events expressinga surprisingly higher average level of insecticidal protein;

gives rise to individual plants, plant tissue, or plant cells expressingan unexpectedly high level of the insecticidal protein; and

gives rise to plants expressing unexpected levels of insecticidalprotein effective in controlling or combating Coleopteran pests andpreventing development of resistance by the Coleopteran pest to theparticular insecticidal protein.

Insecticidal polypeptide refers to a polypeptide having insecticidalproperties, e.g., a polypeptide which exhibits the properties ofinhibiting the growth, development, viability or fecundity of targetinsect pests.

Operably Linked: Nucleic acid or polynucleotide sequences connectedsequentially in linear form, so that the properties of one influence theexpression characteristics of the other. A promoter, for example,operably linked to other polynucleotide sequences (which may consist ofoperator or enhancer sequences, untranslated or translated leadersequences, intron sequences, structural gene coding sequences,non-structural genes, transcription and translation terminationsequences, and polyadenylation sequences) influences the expression of acoding or noncoding sequence, whether the product is RNA, protein, orother product. Similarly, an intron or an untranslated leader sequencecan influence the expression and stability of sequences operably linkedto them, and structural or non-structural gene sequences can beinfluenced by elements operably linked upstream, within, or downstream.

Plant-Expressible Coding Regions: Amino acid coding regions or openreading frames (ORF's) which are expressible in planta because theycontain typical plant regulatory elements facilitating their expression,and often include changes to the coding sequence such that plantpreferred codons are utilized in place of non-preferred codons whereheterologous coding regions are contemplated.

Plastid Transit Peptide: Any amino acid sequence useful in targeting alinked amino acid, such as a protein fusion, to a subcellularcompartment or organelle such as a plastid or chloroplast.

Polynucleotide sequence: Any DNA or RNA sequence of four or moreconsecutive nucleotides or ribonucleotides. Generally polynucleotidesequences as disclosed herein comprise at least 50 or more nucleotidesor ribonucleotides.

Progeny: “Progeny” includes any offspring or descendant of thetransgenic plant, or any subsequent plant which contains thetransgene(s) in operable form. Progeny is not limited to one generation,but rather encompasses the transformant's descendants so long as theycontain or express the transgene(s). Seeds containing transgenic embryosas well as seeds from the transgenic plants and their offspring ordescendants which, after Mendelian segregation continue to contain thetransgene(s), are also important parts of the invention.

Promoter: A recognition site on a DNA sequence or group of DNA sequencesthat provide an expression control element for a preferredpolynucleotide sequence and to which RNA polymerase specifically bindsand initiates RNA synthesis (transcription) of that preferred sequence.

R₀ is the primary regenerant plant derived from transformation of planttissue or cells in culture. Subsequent progeny or generations derivedfrom the R₀ are referred to as R₁ (first generation), R₂ (secondgeneration), etc.

Regeneration: The process of growing a plant from a plant cell or groupof plant cells (e.g., plant protoplast, embryo, callus, or explant).

Structural Coding Sequence refers to a DNA sequence that encodes apeptide, polypeptide, or protein that is made by a cell followingtranscription of the structural coding sequence to messenger RNA (mRNA),followed by translation of the mRNA to the desired peptide, polypeptide,or protein product.

Structural gene: A gene or polynucleotide sequence containing the codingsequence of a desired polypeptide that is expressed by transcription andtranslation to produce the desired polypeptide.

Synthetic gene: Synthetic genes encoding the B. thuringiensisδ-endotoxins of the present invention are those prepared in a mannerinvolving any sort of genetic isolation or manipulation which alters thenaturally occurring coding sequence of the δ-endotoxin gene. Thisincludes isolation of the gene from its naturally occurring state,manipulation of the gene as by codon modification (as described herein),or site-specific mutagenesis (as described herein), truncation of thegene or any other manipulative or isolative method. A synthetic gene canalso be a polynucleotide sequence which is not known to be naturallyoccurring but which encodes a useful polypeptide or other product suchas a tRNA or an antisense polynucleotide. A non-naturally occurringpolynucleotide sequence.

Substantial homology: As this term is used herein, it refers to nucleicacid or polypeptide sequences which are about 86% homologous, to about90% homologous, to about 95% homologous, to about 99% homologous. Morespecifically, the inventors envision substantial homologues to be about86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, and 99 percenthomologous to the referent nucleic acid sequence of polypeptide.

Terminator: With reference to eukaryotic nuclear gene expressionprocesses, the operable 3′ end transcription termination andpolyadenylation sequence. With reference to prokaryotic gene expression,and including plastid or chloroplast gene expression, the operable DNAsequence at the 3′ end of an open reading frame which, for ORF'sexpressing protein product, at least one termination codon in frame withthe coding sequence of the ORF, which may also be followed by a DNAsequence encoding a transcription termination signal which may cause thetranslated RNA or mRNA product to form a hairpin or other threedimensional structure which may or may not act together with one or moresoluble structural proteins to cause transcription to be interrupted.

Transformation: A process of introducing an exogenous polynucleotidesequence (e.g., a vector, or a recombinant or non-recombinant DNA or RNAmolecule) into a cell or protoplast in which that exogenouspolynucleotide is incorporated into a heritable genetic element or iscapable of autonomous replication and thus stably maintained within thatcell or protoplast as well as in the progeny of that cell or protoplast.

Transformed cell: A cell which contains a heritable genetic elementaltered by the introduction of one or more exogenous DNA molecules. Atransgenic cell. Exemplary transformed or transgenic cells include plantcalli derived from a transformed plant cell and particular cells such asleaf, root, stem, e.g., somatic cells, or reproductive (germ) cellsobtained from a transgenic plant.

Transgene: A gene construct, expression cassette, or DNA segment orsequence comprising an ORF which is desired to be expressed in therecipient cell, tissue or organism. This may include an entire plasmid,or other vector, or may simply include the functional coding sequence,region, domain, or segment of the transferred DNA sequence.

Transgenic event: A plant or progeny thereof derived from a plant cellor protoplast manufactured or constructed to contain one or moreexogenous DNA molecules inserted into the nuclear or other genome of theplant cell, or introduced and stably maintained within the cytoplasm ofa plastid, chloroplast, or mitochondria, which confers some physicallydetectable phenotype upon the plant or progeny thereof.

Transgenic plant: A plant or progeny thereof which has been geneticallymodified to contain and express heterologous DNA sequences either asproteins or as nucleic acids. As specifically exemplified herein, atransgenic corn plant is genetically modified to contain and express atleast one heterologous DNA sequence operably linked to and under theregulatory control of transcriptional control sequences which functiontogether in plant cells or tissue or in whole plants to achieveexpression from a nucleic acid sequence encoding an insecticidalδ-endotoxin protein or an amino acid sequence variant thereof. Atransgenic plant may also be referred to as a transformed plant. Atransgenic plant also refers to progeny of the initial transgenic plantwhere those progeny contain and express the heterologous coding sequenceunder the regulatory control of the plant-expressible transcriptioncontrol sequences described herein.

Vector: A polynucleotide capable of replication in a host cell and/or towhich another polynucleotide sequence can be operatively linked so as tobring about replication of the linked sequence. A plasmid is anexemplary vector.

The present invention discloses novel DNA constructs comprisingpolynucleotide sequences encoding B. thuringiensis δ-endotoxins. Methodsfor the construction and expression of synthetic B. thuringiensis genesin plants are well known by those of skill in the art and are describedin detail in U.S. Pat. No. 5,500,365. The present invention contemplatesthe use of Cry3B B. thuringiensis genes in the transformation of bothmonocotyledonous and dicotyledonous plants. To potentiate the expressionof these genes, the present invention provides DNA constructs comprisingpolynucleotide segments encoding plastid targeting peptides positionedupstream of and in frame with the polynucleotide sequences encoding thedesired B. thuringiensis δ-endotoxins, along with various combinationsof untranslated leader sequences, plant functional intron sequences, andtranscription termination and polyadenylation sequences.

In one aspect, nucleotide sequence information provided by the inventionallows for the preparation of relatively short DNA sequences having theability to specifically hybridize to gene sequences of the selectedpolynucleotides disclosed herein. In these aspects, nucleic acid probesof an appropriate length are prepared based on a consideration ofselected polypeptide sequences encoding Coleopteran inhibitory Cry3Bδ-endotoxin polypeptides, e.g., a sequence such as that shown in SEQIDNO:2, SEQID NO:4, SEQID NO:6, SEQID NO:8, SEQID NO:10, and SEQID NO:12.These nucleic acid probes may also be prepared based on a considerationof selected polynucleotide sequences encoding a plastid targetingpeptide, such as those shown in SEQID NO:26 The ability of such nucleicacid probes to specifically hybridize to a gene sequence encoding aδ-endotoxin polypeptide or a plastid targeting peptide sequence lends tothem particular utility in a variety of embodiments. Most importantly,the probes may be used in a variety of assays for detecting the presenceof complementary sequences in a given sample.

In certain embodiments, it is advantageous to use oligonucleotideprimers. The sequence of such primers is designed using a polynucleotideof the present invention for use in detecting, amplifying or mutating adefined segment of a crystal protein gene from B. thuringiensis usingthermal amplification technology. The process may also be used todetect, amplify or mutate a defined segment of the polynucleotideencoding a plastid targeting peptide. Segments of genes related to thepolynucleotides encoding the δ-endotoxin polypeptides and plastidtargeting peptides of the present invention may also be amplified byusing such primers and thermal amplification methods.

To provide certain of the advantages in accordance with the presentinvention, a preferred nucleic acid sequence employed for hybridizationstudies or assays includes a polynucleotide sequences at least about 14to 30 or so nucleotides in length complimentary to a nucleotide sequenceencoding a crystal protein, or polynucleotide sequences at least about14 to 30 or so nucleotides in length complimentary to a nucleotidesequence encoding a plastid targeting peptide.

A size of at least 14 nucleotides in length helps to ensure that thefragment will be of sufficient length to form a duplex molecule that isboth stable and selective. Molecules having complementary sequences oversegments greater than 14 bases in length are generally preferred. Inorder to increase stability and selectivity of the hybrid, and therebyimprove the quality and degree of specific hybrid molecules obtained,one will generally prefer to design nucleic acid molecules havinggene-complementary sequences of 14 to 20 nucleotides, or even longerwhere desired. Such fragments may be readily prepared by, for example,directly synthesizing the fragment by chemical means, by application ofnucleic acid reproduction technology, such as the PCR™ technology ofU.S. Pat. Nos. 4,683,195, and 4,683,202, or by excising selected DNAfragments from recombinant plasmids containing appropriate inserts andsuitable restriction sites.

The present invention also contemplates an expression vector comprisinga polynucleotide of the present invention. Thus, in one embodiment anexpression vector is an isolated and purified DNA molecule comprising apromoter operatively linked to a coding region that encodes apolypeptide of the present invention, which coding region is operativelylinked to a transcription-terminating region, whereby the promoterdrives the transcription of the coding region. The coding region mayinclude a segment encoding a B. thuringiensis δ-endotoxin and a segmentencoding a plastid target peptide. The DNA molecule comprising theexpression vector may also contain a functional intron. As used herein,the terms “operatively linked” or “operably linked” mean that a promoteris connected to a coding region in such a way that the transcription ofthat coding region is controlled and regulated by that promoter. Meansfor operatively linking a promoter to a coding region to regulate bothupstream and downstream are well known in the art.

Preferred plant transformation vectors include those derived from a Tiplasmid of Agrobacterium tumefaciens, as well as those disclosed, e.g.,by Herrera-Estrella (1983), Bevan (1983), Klee (1985) and Eur. Pat Appl.No. EP 0120516.

Promoters that function in bacteria are well known in the art. Exemplaryand preferred promoters for the B. thuringiensis crystal proteinsinclude the sigA, sigE, and sigK gene promoters. Alternatively, native,mutagenized, heterologous, or recombinant promoters derived fromBacillus thuringiensis δ-endotoxin protein coding sequences can be used.

Where an expression vector of the present invention is to be used totransform a plant, a promoter is selected that has the ability to driveexpression in that particular species of plant. Promoters that functionin different plant species are also well known in the art. Promotersuseful in expression of polypeptide coding sequences in plants are thosewhich are inducible, viral, synthetic, or constitutive as described(Paszkowski et al., 1984; Odell et al., 1985), and/or temporallyregulated, spatially regulated, and spatio-temporally regulated (Chau etal., 1989). Preferred promoters include the enhanced CaMV35S promoters,and the FMV35S promoter. Other promoters include the POX promoter, theScbDNA virus early promoter, and the yellow mottle virus promoter.

In accordance with the present invention, expression vectors designed tospecifically potentiate the expression of the polypeptide in thetransformed plant may include certain regions encoding plastid targetingpeptides (PTP). These regions allow for the cellular processes involvedin transcription, translation and expression of the encoded protein tobe fully exploited when associated with certain B. thuringiensisδ-endotoxins. Such plastid targeting peptides function in a variety ofways, such as for example, by transferring the expressed protein to thecell structure in which it most effectively operates, or by transferringthe expressed protein to areas of the cell in which cellular processesnecessary for expression are concentrated.

In the case of Cry3B, elevated expression is critical in obtainingtransgenic corn with CRW control since the LC₅₀ of Cry3B against CRW issignificantly higher than the LC₅₀ of the B. thuringiensis toxinscurrently used to control target pests such as Colorado Potato Beetle inpotato (Cry3A) or European Corn Borer in corn (Cry1Ab).

Increased expression is also especially valuable in that it providesadditional protection against development of resistance via a high dosestrategy (McGaughey and Whalon, 1993; Roush, 1994). High levelexpression is even further desirable as it provides sustained insectprotection in instances where insecticidal gene expression decreases dueto environmental conditions. Additionally and unexpectedly, corn plantstransformed with vectors expressing Coleopteran inhibitory Cry3B orvariant proteins exhibited normal growth and development.

An example of a plastid or chloroplast targeting peptide (CTP) is achloroplast targeting peptide. Chloroplast targeting peptides have beenfound particularly useful in the glyphosate resistant selectable markersystem. In this system, plants transformed to express a proteinconferring glyphosate resistance are transformed with a PTP that targetsthe peptide to the cell's chloroplasts. Glyphosate inhibits the shikimicacid pathway which leads to the biosynthesis of aromatic compoundsincluding amino acids and vitamins. Specifically, glyphosate inhibitsthe conversion of phosphoenolpyruvic acid and 3-phosphoshikimic acid to5-enolpyruvyl-3-phosphoshikimic acid by inhibiting the enzyme5-enolpyruvyl-3-phosphoshikimic acid synthase (EPSP synthase or EPSPS).Supplemental EPSPS, conferred via insertion of a transgene encoding thisenzyme, allows the cell to resist the effects of the glyphosate. Thus,as the herbicide glyphosate functions to kill the cell by interruptingaromatic amino acid biosynthesis, particularly in the cell'schloroplast, the CTP allows increased resistance to the herbicide byconcentrating what glyphosate resistance enzyme the cell expresses inthe chloroplast, ie. in the target organelle of the cell. Exemplaryherbicide resistance enzymes include EPSPS as noted above, glyphosateoxido-reductase (GOX) and the aro-A gene (U.S. Pat. No. 4,535,060).

CTP's can target proteins to chloroplasts and other plastids. Forexample, the target organelle may be the amyloplast. Preferred CTP's ofthe present invention include those targeting both chloroplasts as wellas other plastids. Specific examples of preferred CTP's include themaize RUBISCO SSU protein CTP, and functionally related peptides. Anexemplary CTP polypeptide is shown in SEQ ID NO:26. A polynucleotidesequence encoding for this CTP polypeptide is shown in SEQ ID NO:25.

The expression of a gene which exists in double-stranded DNA forminvolves transcription of messenger RNA (mRNA) from the coding strand ofthe DNA by an RNA polymerase enzyme, and the subsequent processing ofthe mRNA primary transcript inside the nucleus. Transcription of DNAinto mRNA is regulated by a region of DNA usually referred to as the“promoter”. The promoter region contains a sequence of bases thatsignals RNA polymerase to associate with the DNA and to initiate thetranscription of mRNA using one of the DNA strands as a template to makea corresponding strand of RNA. The particular promoter selected shouldbe capable of causing sufficient expression of the enzyme codingsequence to result in the production of an effective insecticidal amountof the B. thuringiensis protein.

The 3′ non-translated region of the chimeric plant genes of the presentinvention also contains a polyadenylation signal which functions inplants to cause the addition of adenylate nucleotides to the 3′ end ofthe RNA. Examples of preferred 3′ regions are (1) the 3′ transcribed,non-translated regions containing the polyadenylation signal ofAgrobacterium tumor-inducing (Ti) plasmid genes, such as the nopalinesynthase (NOS) gene and (2) the 3′ ends of plant genes such as the peassRUBISCO E9 gene (Fischhoff et al., 1987).

A promoter is selected for its ability to direct the transformed plantcell's or transgenic plant's transcriptional activity to the codingregion, to ensure sufficient expression of the enzyme coding sequence toresult in the production of insecticidal amounts of the B. thuringiensisprotein. Structural genes can be driven by a variety of promoters inplant tissues. Promoters can be near-constitutive (i.e. they drivetranscription of the transgene in all tissue), such as the CaMV35Spromoter, or tissue-specific or developmentally specific promotersaffecting dicots or monocots. Where the promoter is a near-constitutivepromoter such as CaMV35S or FMV35S, increases in polypeptide expressionare found in a variety of transformed plant tissues and most plantorgans (e.g., callus, leaf, seed and root). Enhanced or duplicateversions of the CaMV35S and FMV35S promoters are particularly useful inthe practice of this invention (Kay et al., 1987; Rogers, U.S. Pat. No.5,378,619). Tandemly duplicated enhancer sequences have beendemonstrated to be of particular significance, for example, as describedin Neuhaus et al. (Tissue-specific expression from promoter AS-1 intransgenic tobacco. Plant Cell 6: 827-834; 1994).

Those skilled in the art will recognize that there are a number ofpromoters which are active in plant cells, and have been described inthe literature. Such promoters may be obtained from plants or plantviruses and include, but are not limited to, the nopaline synthase (NOS)and octopine synthase (OCS) promoters (which are carried ontumor-inducing plasmids of A. tumefaciens), the cauliflower mosaic virus(CaMV) 19S and 35S promoters, the light-inducible promoter from thesmall subunit of ribulose 1,5-bisphosphate carboxylase (ssRUBISCO, avery abundant plant polypeptide), the rice Act1 promoter, POX promoter,yellow mottle virus promoter, ScBV virus early promoter, the FigwortMosaic Virus (FMV) 35S promoter, and the AS4 35S promoter (root enhancedexpression from 35S promoter linked to multiple tandem as-1 sequences asin Neuhaus et al.). All of these promoters have been used to createvarious types of DNA constructs which have been expressed in plants (seee.g., McElroy et al., 1990, U.S. Pat. No. 5,463,175).

In addition, it may also be preferred to bring about expression of theB. thuringiensis δ-endotoxin in specific tissues of the plant by usingplant integrating vectors containing a tissue-specific promoter.Specific target tissues may include the leaf, stem, root, tuber, seed,fruit, etc., and the promoter chosen should have the desired tissue anddevelopmental specificity. Therefore, promoter function should beoptimized by selecting a promoter with the desired tissue expressioncapabilities and approximate promoter strength and selecting atransformant which produces the desired insecticidal activity in thetarget tissues. This selection approach from the pool of transformantsis routinely employed in expression of heterologous structural genes inplants since there is variation between transformants containing thesame heterologous gene due to the site of gene insertion within theplant genome (commonly referred to as “position effect”). In addition topromoters which are known to cause transcription (constitutive ortissue-specific) of DNA in plant cells, other promoters may beidentified for use in the current invention by screening a plant cDNAlibrary for genes which are selectively or preferably expressed in thetarget tissues and then determine the promoter regions.

An exemplary tissue-specific promoter is the lectin promoter, which isspecific for seed tissue. The lectin protein in soybean seeds is encodedby a single gene (Le1) that is only expressed during seed maturation andaccounts for about 2 to about 5% of total seed mRNA. The lectin gene andseed-specific promoter have been fully characterized and used to directseed specific expression in transgenic tobacco plants (Vodkin et al.,1983; Lindstrom et al., 1990). An expression vector containing a codingregion that encodes a polypeptide of interest can be engineered to beunder control of the lectin promoter and that vector may be introducedinto plants using, for example, a protoplast transformation method (Dhiret al., 1991). The expression of the polypeptide would then be directedspecifically to the seeds of the transgenic plant.

A transgenic plant of the present invention produced from a plant celltransformed with a tissue specific promoter can be crossed with a secondtransgenic plant developed from a plant cell transformed with adifferent tissue specific promoter to produce a hybrid transgenic plantthat shows the effects of transformation in more than one specifictissue.

Other exemplary tissue-specific promoters are corn sucrose synthetase 1(Yang et al., 1990), corn alcohol dehydrogenase 1 (Vogel et al., 1989),corn light harvesting complex (Simpson, 1986), corn heat shock protein(Odell et al., 1985), pea small subunit RuBP carboxylase (Poulsen etal., 1986; Cashmore et al., 1983), Ti plasmid mannopine synthase(McBride and Summerfelt, 1990), Ti plasmid nopaline synthase (Langridgeet al., 1989), petunia chalcone isomerase (Van Tunen et al., 1989), beanglycine rich protein 1 (Keller et al., 1989), CaMV 35s transcript (Odellet al., 1985) and Potato patatin (Wenzler et al., 1989). Preferredpromoters are the cauliflower mosaic virus (CaMV 35S) promoter and theS-E9 small subunit RuBP carboxylase promoter.

The promoters used in the DNA constructs of the present invention may bemodified, if desired, to affect their control characteristics. Forexample, the CaMV35S promoter may be ligated to the portion of thessRUBISCO gene that represses the expression of ssRUBISCO in the absenceof light, to create a promoter which is active in leaves but not inroots. The resulting chimeric promoter may be used as described herein.For purposes of this description, the phrase “CaMV35S” promoter thusincludes variations of CaMV35S promoter, e.g., promoters derived bymeans of ligation with operator regions, random or controlledmutagenesis, etc. Furthermore, the promoters may be altered to containmultiple “enhancer sequences” to assist in elevating gene expression.Examples of such enhancer sequences have been reported by Kay et al.(1987) and Neuhaus et al. (1994).

The RNA produced by a DNA construct of the present invention alsocontains a 5′ non-translated leader sequence. This sequence can bederived from the promoter selected to express the gene, and can bespecifically modified so as to increase translation of the mRNA. The 5′non-translated regions can also be obtained from viral RNAs, fromsuitable eukaryotic genes, or from a synthetic gene sequence. Thepresent invention is not limited to constructs wherein thenon-translated region is derived from the 5′ non-translated sequencethat accompanies the promoter sequence. As shown below, a plant geneleader sequence which is useful in the present invention is the petuniaheat shock protein 70 (hsp70) leader (Winter et al., 1988), the wheatCAB leader, or the wheat PER leader.

An exemplary embodiment of the invention involves the plastid targetingof the B. thuringiensis sequence. Such plastid targeting sequences havebeen isolated from numerous nuclear encoded plant genes and have beenshown to direct importation of cytoplasmically synthesized proteins intoplastids (reviewed in Keegstra and Olsen, 1989). A variety of plastidtargeting sequences, well known in the art, including but not limited toADPGPP, EPSP synthase, or ssRUBISCO, may be utilized in practicing thisinvention. In alternative embodiments preferred, plastidic targetingsequences (peptide and nucleic acid) for monocotyledonous crops mayconsist of a genomic fragment coding containing an intronic sequence aswell as a duplicated proteolytic cleavage site in the encoded plastidictargeting sequences.

The most preferred CTP encoding nucleic acid sequence, referred toherein as zmSSU CTP (SEQ ID NO:25), consisting of a genomic fragmentcontaining an intronic sequence as well as a duplicated proteolyticcleavage site in the encoded plastidic targeting sequences, was derivedfrom plastidic targeting sequence zmS1 (Russell et al., 1993). Directtranslational fusions of zmSSU CTP peptide sequence (SEQ ID NO:26) tothe amino terminus of the sequence has been shown to be useful inobtaining elevated levels of the polypeptide in transgenic maize.In-frame fusions of the zmSSU CTP nucleic acid sequence (SEQ ID NO:25)to a cry3b gene (SEQ ID NO:1) or gene variant can be effected byligation of an NcoI site engineered into the 3′ (C-terminal encoding)end of the zmSSU CTP sequence to a 5′ NcoI site engineered into theN-terminal encoding end of the cry3B or variant coding sequence.

The preferred CTP sequence for dicotyledonous crops consists of agenomic coding fragment containing the chloroplast targeting peptidesequence from the EPSP synthase gene of Arabidopsis thaliana in whichthe transit peptide cleavage site of the pea ssRUBISCO CTP replaces thenative EPSP synthase CTP cleavage site (Klee et al., 1987).

As noted above, the 3′ non-translated region of the chimeric plant genesof the present invention contains a polyadenylation signal whichfunctions in plants to cause the addition of adenylate nucleotides tothe 3′ end of the RNA. Examples of preferred 3′ regions are (1) the 3′transcribed, non-translated regions containing the polyadenylate signalof Agrobacterium tumor-inducing (Ti) plasmid genes, such as the nopalinesynthase (NOS) gene and (2) plant genes such as the pea ssRUBISCO E9gene (Fischhoff et al., 1987).

For optimized expression in monocotyledonous plants, an intron may alsobe included in the DNA expression construct. Such an intron is typicallyplaced near the 5′-end of the mRNA in an untranslated sequence. Thisintron could be obtained from, but not limited to, a set of intronsconsisting of the maize Heat Shock Protein (HSP) 70 intron (U.S. Pat.No. 5,424,412; 1995), the rice Act1 intron (McElroy et al., 1990), theAdh intron 1 (Callis et al., 1987), or the sucrose synthase intron(Vasil et al., 1989). As shown herein, the maize HSP70 intron (SEQIDNO:33) and the rice actin intron (SEQID NO:32) are particularly usefulin the present invention.

RNA polymerase transcribes through a coding DNA sequence to a site wherepolyadenylation occurs. Typically, DNA sequences located a few hundredbase pairs downstream of the polyadenylation site serve to terminatetranscription. Those DNA sequences are referred to herein astranscription-termination regions. Those regions are required forefficient polyadenylation of transcribed messenger RNA (mRNA).

Constructs will typically include the gene of interest along with a 3′end DNA sequence that acts as a signal to terminate transcription andallow for the poly-adenylation of the resultant mRNA. The most preferred3′ elements are contemplated to be those from the nopaline synthase geneof A. tumefaciens (nos 3′end) (Bevan et al., 1983), the terminator forthe T7 transcript from the octopine synthase gene of A. tumefaciens, andthe 3′ end of the protease inhibitor i or ii genes from potato ortomato. Regulatory elements such as TMV Ω element (Gallie, et al.,1989), may further be included where desired.

Another type of element which can regulate gene expression is the DNAsequence between the transcription initiation site and the start of thecoding sequence, termed the untranslated leader sequence. The leadersequence can influence gene expression. Compilations of leader sequenceshave been made to predict optimum or sub-optimum sequences and generate“consensus” and preferred leader sequences (Joshi, 1987). Preferredleader sequences are contemplated to include those which comprisesequences predicted to direct optimum expression of the linkedstructural gene, i.e. to include a preferred consensus leader sequencewhich may increase or maintain mRNA stability and prevent inappropriateinitiation of translation. The choice of such sequences will be known tothose of skill in the art in light of the present disclosure. Sequencesthat are derived from genes that are highly expressed in plants, and inmaize in particular, will be most preferred. One particularly preferredleader may be the wheat CAB leader (SEQID NO:31).

Transcription enhancers or duplications of enhancers could be used toincrease expression. These enhancers often are found 5′ to the start oftranscription in a promoter that functions in eukaryotic cells, but canoften be inserted in the forward or reverse orientation 5′ or 3′ to thecoding sequence. Examples of enhancers include elements from the CaMV35S promoter, octopine synthase genes (Ellis et al., 1987), the riceactin gene, and promoter from non-plant eukaryotes (e.g., yeast; Ma etal., 1988).

The choice of which expression vector and ultimately to which promoter apolypeptide coding region is operatively linked depends directly on thefunctional properties desired, e.g., the location and timing of proteinexpression, and the host cell to be transformed. These are well knownlimitations inherent in the art of constructing recombinant DNAmolecules. However, a vector useful in practicing the present inventionis capable of directing the expression of the polypeptide coding regionto which it is operatively linked.

Typical vectors useful for expression of genes in higher plants are wellknown in the art and include vectors derived from the tumor-inducing(Ti) plasmid of A. tumefaciens described (Rogers et al., 1987). However,several other plant integrating vector systems are known to function inplants including pCaMVCN transfer control vector described (Fromm etal., 1985). pCaMVCN (available from Pharmacia, Piscataway, N.J.)includes the CaMV35S promoter.

In preferred embodiments, the vector used to express the polypeptideincludes a selection marker that is effective in a plant cell,preferably a drug resistance selection marker. One preferred drugresistance marker is the gene whose expression results in kanamycinresistance; i.e. the chimeric gene containing the nopaline synthasepromoter, Tn5 neomycin phosphotransferase II (nptII) and nopalinesynthase 3′ non-translated region described (Rogers et al., 1988).

Means for preparing expression vectors are well known in the art.Expression (transformation) vectors used to transform plants and methodsof making those vectors are described in U.S. Pat. Nos. 4,971,908,4,940,835, 4,769,061 and 4,757,011. Those vectors can be modified toinclude a coding sequence in accordance with the present invention.

A coding region that encodes a polypeptide having the ability to conferinsecticidal activity to a cell is preferably a polynucleotide encodinga B. thuringiensis δ-endotoxin or a functional equivalent of such apolynucleotide. In accordance with such embodiments, a coding regioncomprising the DNA sequences of SEQID NO:1, SEQID NO:3, SEQID NO:5,SEQID NO:7, SEQID NO:9, and SEQID NO:11 are also preferred.

Specific B. thuringiensis δ-endotoxin polypeptide-encoding ORF'scontained within expression cassettes that have been shown to to expressthe B. thuringiensis δ-endotoxins at high levels in transformed plants.Preferred cassettes include those contained in plasmids pMON33709,pMON33710, pMON33722, pMON33723, pMON25096, pMON25097, pMON33741, andpMON33748. The expression cassettes in these plasmids are respectivelyencoded for by the sequences shown in SEQID NO:13, SEQID NO:15, SEQIDNO:36, SEQID NO:38, SEQID NO:17, SEQID NO:19, SEQID NO:21, and SEQIDNO:23. More preferably, plants may be successfully transformed with anyexpression cassettes comprising the nucleotide sequences of nucleotide14 to 3431 of SEQID NO:36, 14 to 3025 of SEQID NO:38, 14 to 3431 ofSEQID NO:17, 14 to 3020 of SEQID NO:19, 14 to 3020 of SEQID NO:21, or 25to 3450 of SEQID NO:23 (pMON33722, pMON33723, pMON25096, pMON25097,pMON33741, and pMON33748). Most preferably, plants may be successfullytransformed with any expression cassettes comprising the nucleotidesequences of nucleotide 14 to 3431 of SEQID NO:17, 14 to 3020 of SEQIDNO:19, 14 to 3020 of SEQID NO:21, or 25 to 3450 of SEQID NO:23(pMON25096, pMON25097, pMON33741, and pMON33748).

The work described herein has identified methods of potentiating inplanta expression of B. thuringiensis δ-endotoxins, which conferresistance to insect pathogens when incorporated into the genome ofsusceptible plants. U.S. Pat. No. 5,500,365 describes a method forsynthesizing plant genes to optimize the expression level of the proteinfor which the synthesized gene encodes. This method relates to themodification of the structural gene sequences of the exogenoustransgene, to make them more “plant-like” and therefore more likely tobe translated and expressed by the plant. A similar method for enhancedexpression of transgenes in monocotyledonous plants is disclosed in U.S.Pat. No. 5,689,052. Agronomic, horticultural, ornamental, and othereconomically or commercially useful plants can be made in accordancewith the methods described herein, to express B. thuringiensisδ-endotoxins at levels high enough to confer resistance to insectpathogens.

Such plants may co-express the B. thuringiensis δ-endotoxin polypeptidealong with other antifungal, antibacterial, or antiviralpathogenesis-related peptides, polypeptides, or proteins; insecticidalproteins; proteins conferring herbicide resistance; and proteinsinvolved in improving the quality of plant products or agronomicperformance of plants. Simultaneous co-expression of multiple proteinsin plants is advantageous in that it exploits more than one mode ofaction to control plant pathogenic damage. This can minimize thepossibility of developing resistant pathogen strains, broaden the scopeof resistance, and potentially result in a synergistic insecticidaleffect, thereby enhancing plants ability to resist insect infestation(WO 92/17591).

Ultimately, the most desirable DNA segments for introduction into amonocot genome may be homologous genes or gene families which encode adesired trait (for example, increased yield), and which are introducedunder the control of novel promoters or enhancers, etc., or perhaps evenhomologous or tissue specific (e.g., root-collar/sheath-, whorl-,stalk-, earshank-, kernel- or leaf-specific) promoters or controlelements. Indeed, it is envisioned that a particular use of the presentinvention may be the production of transformants comprising a transgenewhich is targeted in a tissue-specific manner. For example, insectresistant genes may be expressed specifically in the whorl andcollar/sheath tissues which are targets for the first and second broods,respectively, of ECB. Likewise, it is desireable that genes encodingproteins with particular activity against rootworm be preferentiallyexpressed in root tissues.

Vectors for use in tissue-specific targeting of gene expression intransgenic plants typically will include tissue-specific promoters andalso may include other tissue-specific control elements such as enhancersequences. Promoters which direct specific or enhanced expression incertain plant tissues will be known to those of skill in the art inlight of the present disclosure.

It also is contemplated that tissue specific expression may befunctionally accomplished by introducing a constitutively expressed gene(all tissues) in combination with an antisense gene that is expressedonly in those tissues where the gene product is not desired. Forexample, a gene coding for the crystal toxin protein from B.thuringiensis may be introduced such that it is expressed in all tissuesusing the 35S promoter from Cauliflower Mosaic Virus. Alternatively, arice actin promoter or a histone promoter from a dicot or monocotspecies also could be used for constitutive expression of a gene.Furthermore, it is contemplated that promoters combining elements frommore than one promoter may be useful. For example, U.S. Pat. No.5,491,288 discloses combining a Cauliflower Mosaic Virus promoter with ahistone promoter. Therefore, expression of an antisense transcript of aBt δ-endotoxin gene in a maize kernel, using for example a zeinpromoter, would prevent accumulation of the δ-endotoxin in seed. Hencethe protein encoded by the introduced gene would be present in alltissues except the kernel. It is specifically contemplated by theinventors that a similar strategy could be used with the instantinvention to direct expression of a screenable or selectable marker inseed tissue.

Alternatively, one may wish to obtain novel tissue-specific promotersequences for use in accordance with the present invention. To achievethis, one may first isolate cDNA clones from the tissue concerned andidentify those clones which are expressed specifically in that tissue,for example, using Northern blotting. Ideally, one would like toidentify a gene that is not present in a high copy number, but whichgene product is relatively abundant in specific tissues. The promoterand control elements of corresponding genomic clones may thus belocalized using the techniques of molecular biology known to those ofskill in the art.

It is contemplated that expression of some genes in transgenic plantswill be desired only under specified conditions. For example, it isproposed that expression of certain genes that confer resistance toenvironmentally stress factors such as drought will be desired onlyunder actual stress conditions. It further is contemplated thatexpression of such genes throughout a plants development may havedetrimental effects. It is known that a large number of genes exist thatrespond to the environment. For example, expression of some genes suchas rbcS, encoding the small subunit of ribulose bisphosphatecarboxylase, is regulated by light as mediated through phytochrome.Other genes are induced by secondary stimuli. For example, synthesis ofabscisic acid (ABA) is induced by certain environmental factors,including but not limited to water stress. A number of genes have beenshown to be induced by ABA (Skriver and Mundy, 1990). It also isexpected that expression of genes conferring resistance to insectpredation would be desired only under conditions of actual insectinfestation. Therefore, for some desired traits, inducible expression ofgenes in transgenic plants will be desired.

It is proposed that, in some embodiments of the present invention,expression of a gene in a transgenic plant will be desired only in acertain time period during the development of the plant. Developmentaltiming frequently is correlated with tissue specific gene expression.For example expression of zein storage proteins is initiated in theendosperm about 15 days after pollination.

It is contemplated that the method described in this invention could beused to obtain substantially improved expression of a number of novel B.thuringiensis endotoxins isolated as described below. Identification ofnew Bacillus thuringiensis strains encoding crystalline endotoxins withinsecticidal activity has been described previously (Donovan et al.,1992). Isolation of the B. thuringiensis endotoxin, followed by aminoterminal amino acid sequencing, back-translation of the amino acidsequence to design an oligonucleotide probe or use of a related B.thuringiensis gene as a probe, followed by cloning of the gene encodingthe endotoxin by hybridization are familiar to those skilled in the artand have been described (see e.g., Donovan et al., 1992, U.S. Pat. No.5,264,364). Cry3Bb Bacillus thuringiensis δ-endotoxins with improvedColeopteran inhibitory activity can be achieved using the methodsdescribed in English et al. (WO 99/31248).

A plant transformed with an expression vector of the present inventionis also contemplated. A transgenic plant derived from such a transformedor transgenic cell is also contemplated. Those skilled in the art willrecognize that a chimeric plant gene containing a structural codingsequence of the present invention can be inserted into the genome of aplant by methods well known in the art. Such methods for DNAtransformation of plant cells include Agrobacterium-mediated planttransformation, the use of liposomes, transformation using viruses orpollen, electroporation, protoplast transformation, gene transfer intopollen, injection or vacuum infiltration (Bechtold et al., Meth. Mo.Biol., 82:259-266; 1998) into reproductive organs, injection intoimmature embryos and particle bombardment. Each of these methods hasdistinct advantages and disadvantages. Thus, one particular method ofintroducing genes into a particular plant strain may not necessarily bethe most effective for another plant strain, but it is well known whichmethods are useful for a particular plant strain.

Technology for introduction of DNA into cells is well-known to those ofskill in the art. Four general methods for delivering a gene into cellshave been described: (1) chemical methods (Graham and van der Eb, 1973);(2) physical methods such as microinjection (Capecchi, 1980),electroporation (Wong and Neumann, 1982; Fromm et al., 1985) and thegene gun (Johnston and Tang, 1994; Fynan et al., 1993); (3) viralvectors (Clapp, 1993; Lu et al., 1993; Eglitis and Anderson, 1988a;1988b); and (4) receptor-mediated mechanisms (Curiel et al., 1991; 1992;Wagner et al., 1992).

An advantageous method for delivering transforming DNA segments to plantcells is microprojectile bombardment. In this method, particles may becoated with nucleic acids and delivered into cells by a propellingforce. Exemplary particles include those comprised of tungsten, gold,platinum, and the like. Using these particles, DNA is carried throughthe cell wall and into the cytoplasm on the surface of small metalparticles as described (Klein et al., 1987; Klein et al., 1988). Themetal particles penetrate through several layers of cells and thus allowthe transformation of cells within tissue explants.

An advantage of microprojectile bombardment, in addition to it being aneffective means of reproducibly stably transforming plant cells, is thatneither the isolation of protoplasts (Cristou et al., 1988) nor thesusceptibility to Agrobacterium infection is required. An illustrativeembodiment of a method for delivering DNA into plant cells byacceleration is a Biolistics Particle Delivery System, which can be usedto propel particles coated with DNA or cells through a screen, such as astainless steel or Nytex screen, onto a filter surface covered with theplant cultured cells in suspension. The screen disperses the particlesso that they are not delivered to the recipient cells in largeaggregates. It is believed that a screen intervening between theprojectile apparatus and the cells to be bombarded reduces the size ofprojectiles aggregate and may contribute to a higher frequency oftransformation by reducing damage inflicted on the recipient cells byprojectiles that are too large.

For the bombardment, cells in suspension are preferably concentrated onfilters or solid culture medium. Alternatively, immature embryos orother target cells may be arranged on solid culture medium. The cells tobe bombarded are positioned at an appropriate distance below themacroprojectile stopping plate. If desired, one or more screens are alsopositioned between the acceleration device and the cells to bebombarded. Through the use of techniques set forth herein one may obtainup to 1000 or more foci of cells transiently expressing a marker gene.The number of cells in a focus which express the exogenous gene product48 hours post-bombardment often range from 1 to 10 and average 1 to 3.

In bombardment transformation, one may optimize the pre-bombardmentculturing conditions and the bombardment parameters to yield the maximumnumbers of stable transformants. Both the physical and biologicalparameters for bombardment are important in this technology. Physicalfactors are those that involve manipulating the DNA/microprojectileprecipitate or those that affect the flight and velocity of either themacro- or microprojectiles. Biological factors include all stepsinvolved in manipulation of cells before and immediately afterbombardment, the osmotic adjustment of target cells to help alleviatethe trauma associated with bombardment, and also the nature of thetransforming DNA, such as linearized DNA or intact supercoiled plasmids.It is believed that pre-bombardment manipulations are especiallyimportant for successful transformation of immature plant embryos.

Accordingly, it is contemplated that one may desire to adjust various ofthe bombardment parameters in small scale studies to fully optimize theconditions. One may particularly wish to adjust physical parameters suchas gap distance, flight distance, tissue distance, and helium pressure.One may also minimize the trauma reduction factors (TRFs) by modifyingconditions which influence the physiological state of the recipientcells and which may therefore influence transformation and integrationefficiencies. For example, the osmotic state, tissue hydration and thesubculture stage or cell cycle of the recipient cells may be adjustedfor optimum transformation. The execution of other routine adjustmentswill be known to those of skill in the art in light of the presentdisclosure.

The methods of particle-mediated transformation is well-known to thoseof skill in the art. U.S. Pat. No. 5,015,580 describes thetransformation of soybeans using such a technique.

Agrobacterium-mediated transfer is a widely applicable system forintroducing genes into plant cells because the DNA can be introducedinto whole plant tissues, thereby bypassing the need for regeneration ofan intact plant from a protoplast. The use of Agrobacterium-mediatedplant integrating vectors to introduce DNA into plant cells is wellknown in the art. See, for example, the methods described (Fraley etal., 1985; Rogers et al., 1987). The genetic engineering of cottonplants using Agrobacterium-mediated transfer is described in U.S. Pat.No. 5,004,863; like transformation of lettuce plants is described inU.S. Pat. No. 5,349,124; and the Agrobacterium-mediated transformationof soybean is described in U.S. Pat. No. 5,416,011. Further, theintegration of the Ti-DNA is a relatively precise process resulting infew rearrangements. The region of DNA to be transferred is defined bythe border sequences, and intervening DNA is usually inserted into theplant genome as described (Spielmann et al., 1986; Jorgensen et al.,1987).

Modern Agrobacterium transformation vectors are capable of replicationin E. coli as well as Agrobacterium, allowing for convenientmanipulations as described (Klee et al., 1985). Moreover, recenttechnological advances in vectors for Agrobacterium-mediated genetransfer have improved the arrangement of genes and restriction sites inthe vectors to facilitate construction of vectors capable of expressingvarious polypeptide coding genes. The vectors described (Rogers et al.,1987), have convenient multi-linker regions flanked by a promoter and apolyadenylation site for direct expression of inserted polypeptidecoding genes and are suitable for present purposes. In addition,Agrobacterium containing both armed and disarmed Ti genes can be usedfor the transformations. In those plant varieties whereAgrobacterium-mediated transformation is efficient, it is the method ofchoice because of the facile and defined nature of the gene transfer.

Agrobacterium-mediated transformation of leaf disks and other tissuessuch as cotyledons and hypocotyls appears to be limited to plants thatAgrobacterium naturally infects. Agrobacterium-mediated transformationis most efficient in dicotyledonous plants. Few monocots appear to benatural hosts for Agrobacterium, although transgenic plants have beenproduced in asparagus using Agrobacterium vectors as described (Bytebieret al., 1987). Other monocots recently have also been transformed withAgrobacterium. Included in this group are corn (Ishida et al.) and rice(Cheng et al.).

A transgenic plant formed using Agrobacterium transformation methodstypically contains a single gene on one chromosome. Such transgenicplants can be referred to as being heterozygous for the added gene.However, inasmuch as use of the word “heterozygous” usually implies thepresence of a complementary gene at the same locus of the secondchromosome of a pair of chromosomes, and there is no such gene in aplant containing one added gene as here, it is believed that a moreaccurate name for such a plant is an independent segregant, because theadded, exogenous gene segregates independently during mitosis andmeiosis.

An independent segregant may be preferred when the plant iscommercialized as a hybrid, such as corn. In this case, an independentsegregant containing the gene is crossed with another plant, to form ahybrid plant that is heterozygous for the gene of interest.

An alternate preference is for a transgenic plant that is homozygous forthe added structural gene; i.e. a transgenic plant that contains twoadded genes, one gene at the same locus on each chromosome of achromosome pair. A homozygous transgenic plant can be obtained bysexually mating (selling) an independent segregant transgenic plant thatcontains a single added gene, germinating some of the seed produced andanalyzing the resulting plants produced for gene of interest activityand mendelian inheritance indicating homozygosity relative to a control(native, non-transgenic) or an independent segregant transgenic plant.

Two different transgenic plants can be mated to produce offspring thatcontain two independently segregating added, exogenous genes. Selfing ofappropriate progeny can produce plants that are homozygous for bothadded, exogenous genes that encode a polypeptide of interest.Back-crossing to a parental plant and out-crossing with a non-transgenicplant are also contemplated.

Transformation of plant protoplasts can be achieved using methods basedon calcium phosphate precipitation, polyethylene glycol treatment,electroporation, and combinations of these treatments (see e.g.,Potrykus et al., 1985; Lorz et al., 1985; Fromm et al., 1985; Uchimiyaet al., 1986; Callis et al., 1987; Marcotte et al., 1988). Applicationof these systems to different plant germplasm depends upon the abilityto regenerate that particular plant variety from protoplasts.Illustrative methods for the regeneration of cereals from protoplastsare described (see, e.g., Toriyama et al., 1986; Yamada et al., 1986;Abdullah et al., 1986). To transform plant germplasm that cannot besuccessfully regenerated from protoplasts, other ways to introduce DNAinto intact cells or tissues can be utilized. For example, regenerationof cereals from immature embryos or explants can be effected asdescribed (Vasil, 1988). DNA can also be introduced into plants bydirect DNA transfer into pollen as described (Hess, 1987). Expression ofpolypeptide coding genes can be obtained by injection of the DNA intoreproductive organs of a plant as described (Pena et al., 1987). DNA canalso be injected directly into the cells of immature embryos and therehydration of desiccated embryos as described (Neuhaus et al., 1987;Benbrook et al., 1986).

Unmodified bacterial genes are often poorly expressed in transgenicplant cells. Several reports have disclosed methods for improvingexpression of recombinant genes in plants (Murray et al., 1989; Diehn etal., 1996; Iannacone et al., 1997; Rouwendal et al., 1997; Futterer etal., 1997; and Futterer and Hohn, 1996). These reports disclose variousmethods for engineering coding sequences to represent sequences whichare more efficiently translated based on plant codon frequency tables,improvements in codon third base position bias, using recombinantsequences which avoid suspect polyadenylation or A/F rich domains orintron splicing consensus sequences. While these methods for syntheticgene construction are notable, synthetic genes of the present inventionwere prepared according to the method of Brown et al. (U.S. Pat. No.5,689,052; 1997). Thus, the present invention provides a method forpreparing synthetic plant genes express in planta a desired proteinproduct at levels significantly higher than the wild-type genes.Briefly, according to Brown et al., the frequency of rare and semi-raremonocotyledonous codons in a polynucleotide sequence encoding a desiredprotein are reduced and replaced with more preferred monocotyledonouscodons. Enhanced accumulation of a desired polypeptide encoded by amodified polynucleotide sequence in a monocotyledonous plant is theresult of increasing the frequency of preferred codons by analyzing thecoding sequence in successive six nucleotide fragments and altering thesequence based on the frequency of appearance of the six-mers as to thefrequency of appearance of the rarest 284, 484, and 664 six-mers inmonocotyledenous plants. Furthermore, Brown et al. disclose the enhancedexpression of a recombinant gene by applying the method for reducing thefrequency of rare codons with methods for reducing the occurrence ofpolyadenylation signals and intron splice sites in the nucleotidesequence, removing self-complementary sequences in the nucleotidesequence and replacing such sequences with nonself-complementarynucleotides while maintaining a structural gene encoding thepolypeptide, and reducing the frequency of occurrence of 5′-CG-3′dinucleotide pairs in the nucleotide sequence. These steps are performedsequentially and have a cumulative effect resulting in a nucleotidesequence containing a preferential utilization of the more-preferredmonocotyledonous codons for monocotyledonous plants for a majority ofthe amino acids present in the desired polypeptide.

Thus, the amount of a gene coding for a polypeptide of interest (i.e. abacterial crystal protein or δ-endotoxin polypeptide or such δ-endotoxinlinked to a plastid targeting peptide) can be increased in plants bytransforming those plants using transformation methods such as thosedisclosed herein.

After effecting delivery of exogenous DNA to recipient cells, the nextstep to obtain a transgenic plant generally concern identifying thetransformed cells for further culturing and plant regeneration. Asmentioned herein, in order to improve the ability to identifytransformants, it is preferable to employ a selectable or screenablemarker gene as, or in addition to, the expressible gene of interest. Inthis case, one would then generally assay the potentially transformedcell population by exposing the cells to a selective agent or agents, orone would screen the cells for the desired marker gene trait.

An exemplary embodiment of methods for identifying transformed cellsinvolves exposing the transformed cultures to a selective agent, such asa metabolic inhibitor, an antibiotic, herbicide or the like. Cells whichhave been transformed and have stably integrated a marker geneconferring resistance to the selective agent used, will grow and dividein culture. Sensitive cells will not be amenable to further culturing.One example of a preferred marker gene encoding an EPSPS synthase whichis resistant to glyphosate inhibition. When this gene is used as aselectable marker, the putatively transformed cell culture is treatedwith glyphosate. Upon treatment, transgenic cells will be available forfurther culturing while sensitive, or non-transformed cells, will not.This method is described in detail in U.S. Pat. No. 5,569,834. Anotherexample of a preferred selectable marker system is the nptII system bywhich resistance to the antibiotics kanamycin, neomycin, and paromomycinor related antibiotics is conferred, as described in U.S. Pat. No.5,569,834. Again, after transformation with this system transformedcells containing a plant expressible nptII gene will be available forfurther culturing upon treatment with kanamycin or related antibiotic,while non-transformed cells will not. Use of this type of a selectablemarker system is described in Brown et al. (U.S. Pat. No. 5,424,412).Another screenable marker which may be used is the gene coding for greenfluorescent protein. All contemplated assays are nondestructive andtransformed cells may be cultured further following identification.

It is further contemplated that combinations of screenable andselectable markers will be useful for identification of transformedcells. In some cell or tissue types a selection agent, such asglyphosate or kanamycin, may either not provide enough killing activityto clearly recognize transformed cells or may cause substantialnonselective inhibition of transformants and non-transformants alike,thus causing the selection technique to not be effective. It is proposedthat selection with a growth inhibiting compound, such as glyphosate atconcentrations below those that cause 100% inhibition followed byscreening of growing tissue for expression of a screenable marker genesuch as kanamycin would allow one to recover transformants from cell ortissue types that are not amenable to selection alone. It is proposedthat combinations of selection and screening may enable one to identifytransformants in a wider variety of cell and tissue types.

The development or regeneration of plants from either single plantprotoplasts or various explants is well known in the art (Weissbach andWeissbach, 1988). This regeneration and growth process typicallyincludes the steps of selection of transformed cells, culturing thoseindividualized cells through the usual stages of embryonic developmentthrough the rooted plantlet stage. Transgenic embryos and seeds aresimilarly regenerated. The resulting transgenic rooted shoots arethereafter planted in an appropriate plant growth medium such as soil.

The development or regeneration of plants containing the foreign,exogenous gene that encodes a polypeptide of interest introduced byAgrobacterium from leaf explants can be achieved by methods well knownin the art such as described (Horsch et al., 1985). In this procedure,transformants are cultured in the presence of a selection agent and in amedium that induces the regeneration of shoots in the plant strain beingtransformed as described (Fraley et al., 1983). In particular, U.S. Pat.No. 5,349,124 details the creation of genetically transformed lettucecells and plants resulting therefrom which express hybrid crystalproteins conferring insecticidal activity against Lepidopteran larvae tosuch plants. This procedure typically produces shoots within two to fourmonths and those shoots are then transferred to an appropriateroot-inducing medium containing the selective agent and an antibiotic toprevent bacterial growth. Shoots that rooted in the presence of theselective agent to form plantlets are then transplanted to soil or othermedia to allow the production of roots. These procedures vary dependingupon the particular plant strain employed, such variations being wellknown in the art.

A transgenic plant of this invention thus has an increased amount of acoding region encoding a B. thuringiensis δ-endotoxin polypeptide orvariant thereof or may encode such a δ-endotoxin linked to a plastidtargeting peptide. A preferred transgenic plant is an independentsegregant and can transmit that gene and its activity to its progeny. Amore preferred transgenic plant is homozygous for that gene, andtransmits that gene to all of its offspring on sexual mating. Seed froma transgenic plant may be grown in the field or greenhouse, andresulting sexually mature transgenic plants are self-pollinated togenerate true breeding plants. The progeny from these plants become truebreeding lines that are evaluated for increased expression of thetransgene encoding the δ-endotoxin.

To identify a transgenic plant expressing high levels of the δ-endotoxinof interest, it is necessary to screen the herbicide or antibioticresistant transgenic, regenerated plants (R₀ generation) forinsecticidal activity and/or expression of the gene of interest. Thiscan be accomplished by various methods well known to those skilled inthe art, including but not limited to: 1) obtaining small tissue samplesfrom the transgenic R₀ plant and directly assaying the tissue foractivity against susceptible insects in parallel with tissue derivedfrom a non-expressing, negative control plant. For example, R₀transgenic corn plants expressing B. thuringiensis endotoxins such asCry3B can be identified by assaying leaf tissue or root tissue derivedfrom such plants for activity against CRW; 2) analysis of proteinextracts by enzyme linked immunoassays (ELISAs) specific for the gene ofinterest (Cry3B); or 3) reverse transcriptase thermal amplification toidentify events expressing the gene of interest.

The genes and δ-endotoxins according to the subject invention includenot only the full length sequences disclosed herein but also fragmentsof these sequences, or fusion proteins, which retain the characteristicinsecticidal activity of the sequences specifically exemplified herein.

It should be apparent to a person of skill in the art that insecticidalδ-endotoxins can be identified and obtained through several means. Thespecific genes, or portions thereof, may be obtained from a culturedepository, or constructed synthetically, for example, by use of a genemachine. Variations of these genes may be readily constructed usingstandard techniques for making point mutations. Also, fragments of thesegenes can be made using commercially available exonucleases orendonucleases according to standard procedures. For example, enzymessuch as Bal31 or site-directed mutagenesis can be used to systematicallycut off nucleotides from the ends of these genes. Also, genes which codefor active fragments may be obtained using a variety of otherrestriction enzymes. Proteases may be used to directly obtain activefragments of these δ-endotoxins.

Equivalent δ-endotoxins and/or genes encoding these δ-endotoxins canalso be isolated from Bacillus strains and/or DNA libraries using theteachings provided herein. For example, antibodies to the δ-endotoxinsdisclosed and claimed herein can be used to identify and isolate otherδ-endotoxins from a mixture of proteins. Specifically, antibodies may beraised to the portions of the δ-endotoxins which are most constant andmost distinct from other B. thuringiensis δ-endotoxins. These antibodiescan then be used to specifically identify equivalent δ-endotoxins withthe characteristic insecticidal activity by immunoprecipitation, enzymelinked immunoassay (ELISA), or Western blotting.

A further method for identifying the δ-endotoxins and genes of thesubject invention is through the use of oligonucleotide probes. Theseprobes are nucleotide sequences having a detectable label. As is wellknown in the art, if the probe molecule and nucleic acid samplehybridize when together in a sample by forming hydrogen bonds betweenthe two molecules, it can be reasonably assumed that the probe andsample are essentially identical or substantially similar or homologousat least along the length of the probe. The probe's detectable labelprovides a means for determining in a known manner whether hybridizationhas occurred. Such a probe analysis provides a rapid method foridentifying insecticidal δ-endotoxin genes of the subject invention.

Duplex formation and stability depend on substantial complementarybetween the two strands of a hybrid, and, as noted above, a certaindegree of mismatch can be tolerated. Therefore, the probes of thesubject invention include mutations (both single and multiple),deletions, insertions of the described sequences, and combinationsthereof, wherein said mutations, insertions and deletions permitformation of stable hybrids with the target polynucleotide of interest.Mutations, insertions, and deletions can be produced in a givenpolynucleotide sequence in many ways, by methods currently known to anordinarily skilled artisan, and perhaps by other methods which maybecome known in the future.

The potential variations in the probes listed is due, in part, to theredundancy of the genetic code. Because of the redundancy of the geneticcode, more than one coding nucleotide triplet (codon) can be used formost of the amino acids used to make proteins. Therefore differentnucleotide sequences can code for a particular amino acid. Thus, theamino acid sequences of the B. thuringiensis δ-endotoxins and peptides,and the plastid targeting peptides and the polynucleotides which codefor them, can be prepared by equivalent nucleotide sequences encodingthe same amino acid sequence of the protein or peptide.

Site-specific mutagenesis is a technique useful in the preparation ofindividual peptides, or biologically functional equivalent proteins orpeptides, through specific mutagenesis of the underlying DNA. Thetechnique further provides a ready ability to prepare and test sequencevariants, for example, incorporating one or more of the foregoingconsiderations, by introducing one or more nucleotide sequence changesinto the DNA.

In general, the technique of site-specific mutagenesis is well known inthe art, as exemplified by various publications. As will be appreciated,the technique typically employs a phage vector which exists in both asingle stranded and double stranded form. Typical vectors useful insite-directed mutagenesis include vectors such as the M13 phage orplasmids containing an M13 origin of replication. These phage arereadily commercially available and their use is generally well known tothose skilled in the art.

Modification and changes may be made in the structure of the peptides ofthe present invention and DNA segments which encode them and stillobtain a functional molecule that encodes a protein or peptide withdesirable characteristics. The biologically functional equivalentpeptides, polypeptides, and proteins contemplated herein should possessabout 80% or greater sequence similarity, preferably about 85% orgreater sequence similarity, and most preferably about 90% or greatersequence similarity, to the sequence of, or corresponding moiety within,the fundamental Cry3B amino acid sequence.

The following is a discussion based upon changing the amino acids of aprotein to create an equivalent, or even an improved, second-generationmolecule. In particular embodiments of the invention, mutated crystalproteins are contemplated to be useful for increasing the insecticidalactivity of the protein, and consequently increasing the insecticidalactivity and/or expression of the recombinant transgene in a plant cell.The amino acid changes may be achieved by changing the codons of the DNAsequence, according to the codons given in readily available amino acidcodon tables.

For example, certain amino acids may be substituted for other aminoacids in a protein structure without appreciable loss of interactivebinding capacity with structures such as, for example, antigen-bindingregions of antibodies or binding sites on substrate molecules. Since itis the interactive capacity and nature of a protein that defines thatprotein's biological functional activity, certain amino acid sequencesubstitutions can be made in a protein sequence, and, of course, itsunderlying DNA coding sequence, and nevertheless obtain a protein withlike properties. It is thus contemplated by the inventors that variouschanges may be made in the peptide sequences of the disclosedcompositions, or corresponding DNA sequences which encode said peptideswithout appreciable loss of their biological utility or activity.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, 1982, incorporate herein byreference). It is accepted that the relative hydropathic character ofthe amino acid contributes to the secondary structure of the resultantprotein, which in turn defines the interaction of the protein with othermolecules, for example, enzymes, substrates, receptors, DNA, antibodies,antigens, and the like.

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e. still obtaina biological functionally equivalent protein. It is also understood inthe art that the substitution of like amino acids can be madeeffectively on the basis of hydrophilicity. U.S. Pat. No. 4,554,101states that the greatest local average hydrophilicity of a protein, asgoverned by the hydrophilicity of its adjacent amino acids, correlateswith a biological property of the protein. It is understood that anamino acid can be substituted for another having a similarhydrophilicity value and still obtain a biologically equivalent, and inparticular, an immunologically equivalent protein.

As outlined above, amino acid substitutions are generally thereforebased on the relative similarity of the amino acid side-chainsubstituents, for example, their hydrophobicity, hydrophilicity, charge,size, and the like. Exemplary substitutions which take various of theforegoing characteristics into consideration are well known to those ofskill in the art and include: arginine and lysine; glutamate andaspartate; serine and threonine; glutamine and asparagine; and valine,leucine and isoleucine.

Polynucleotides encoding δ-endotoxins derived from B. thuringiensis areknown by those skilled in the art, to be poorly expressed whenincorporated into the nuclear DNA of transgenic plants (reviewed byDiehn et al., 1996). Preferably, a nucleotide sequence encoding theδ-endotoxin of interest is designed essentially as described in U.S.Pat. Nos. 5,500,365 and 5,689,052. Examples of nucleotide sequencesuseful for expression include but are not limited to, cry3B (SEQIDNO:5), cry3Bb1 (SEQID NO:1), cry3Bb2 (SEQID NO:3), v11231 (SEQID NO:7),11231mv1 (SEQID NO:9), and 11231mv2 (SEQID NO:11).

Peptides, polypeptides, and proteins biologically functionallyequivalent to Cry3B include amino acid sequences containing conservativeamino acid changes in the fundamental sequence shown in SEQID NO:2,SEQID NO:4, SEQID NO:8, SEQID NO:10, and SEQID NO:12 (Cry3Bb1, Cry3Bb2,v11231, 11231mv1, 11231mv2, Cry3Bb.11231, or Cry3Bb.11098, etc). In suchamino acid sequences, one or more amino acids in the fundamentalsequence is (are) substituted with another amino acid(s), the charge andpolarity of which is similar to that of the native amino acid, i.e. aconservative amino acid substitution, resulting in a silent change.

Substitutes for an amino acid within the fundamental polypeptidesequence can be selected from other members of the class to which thenaturally occurring amino acid belongs. Amino acids can be divided intothe following four groups: (1) acidic amino acids; (2) basic aminoacids; (3) neutral polar amino acids; and (4) neutral non-polar aminoacids. Representative amino acids within these various groups include,but are not limited to: (1) acidic (negatively charged) amino acids suchas aspartic acid and glutamic acid; (2) basic (positively charged) aminoacids such as arginine, histidine, and lysine; (3) neutral polar aminoacids such as glycine, serine, threonine, cyteine, cystine, tyrosine,asparagine, and glutamine; (4) neutral nonpolar (hydrophobic) aminoacids such as alanine, leucine, isoleucine, valine, proline,phenylalanine, tryptophan, and methionine.

Conservative amino acid changes within the fundamental polypeptidesequence can be made by substituting one amino acid within one of thesegroups with another amino acid within the same group. Biologicallyfunctional equivalents of Cry3B can have 10 or fewer conservative aminoacid changes, more preferably seven or fewer conservative amino acidchanges, and most preferably five or fewer conservative amino acidchanges. The encoding nucleotide sequence (gene, plasmid DNA, cDNA,non-naturally occurring, or synthetic DNA) will thus have correspondingbase substitutions, permitting it to encode biologically functionalequivalent forms of Cry3B.

The present invention provides methods and compositions for expressingColeopteran inhibitory Cry3B B. thuringiensis δ-endotoxins or amino acidsequence variants thereof at unexpectedly high levels in transgenicplants. The disclosed methods and compositions may exploit any of theDNA constructs disclosed as well as any of the transformation vectorsdisclosed herein. The contemplated methods and compositions enableCry3Bb δ-endotoxins or amino acid sequence variants thereof to beexpressed in plants without negatively affecting the recovery ofagronomic qualities of transgenic plants. The inventions describedherein also enables expression of Cry3B δ-endotoxins and variants atlevels up to 500 times higher than that achieved by previous methods andcompositions.

The methods described here thus enables plants expressing Cry3B orvariants to be used as either an alternative or supplement to plantsexpressing other Cry proteins such as a Cry3B variant, a Cry3A or Cry3Dor variant, CryET33 and CryET34 or variants thereof, a CryET70 orvariant, a CryET29 or variant, a Cry6A or Cry6B or variant, a Cry8B orvariant, insecticidal acyl lipid hydrolases, combinations of amino acidoxidases and tedanalactam synthases, and other insecticidal proteinssuch as VIP 1 and VIP3 and various combinations isolated fromHeterorhabdus, Photorhabdus, and Xenorhabdus species for both controland resistance management of key insect pests, including Ostrina sp,Diatraea sp, Diabrotica, Helicoverpa sp, Spodoptera sp in Zea mays;Heliothis virescens, Helicoverpa sp, Pectinophora sp. in Gossypiumhirsutum; and Anticarsia sp, Pseudoplusia sp, Epinotia sp in Glycinemax. It is also contemplated that the methods described may be used todramatically increase expression of B. thuringiensis δ-endotoxinsincluding and related to Cry3, thus increasing its effectiveness againsttarget pests and decreasing the likelihood of evolved resistance tothese proteins. In one embodiment of the present invention, a Cry3δ-endotoxin is expressed. Target pests of this protein and their commonhosts are shown below in Table 1.

TABLE 1 Target Pests Affected by Coleopteran Active (Inhibitory) Cry3Bδ-Endotoxin and Common Plant Hosts of Those Pests Pests HostsLeptinotarsa decemlineata Potato (Colorado Potato Beetle) Diabroticabarberi Corn (Northern Corn Rootworm) Diabrotica undecimpunctata Corn(Southern Corn Rootworm) Diabrotica virgifera Corn (Western CornRootworm) Anthonomis grandis Cotton (Boll Weevil) Triboleum castaneumWheat (Red Flour Beetle) Popilla japonica Wheat (Japanese Flour Beetle)

Antibodies were required for studies comparing expression of variousCry3 coding sequences, so polyclonal serum was generated as follows.Cry3 Bt crystals were collected from a sporulated fermentation ofBacillus thuringiensis recombinant strain 11037 expressing nativeCry3Bb. Crystals were solubilized in 100 mM sodium carbonate buffer,pH10.5, to give a concentration of 2.7 mg protein per mL as measured bya colorimetric bicinchoninic acid assay (Smith et al, 1985). A samplewas diluted to a concentration of 0.4 mg/mL and mixed with an equalvolume of Freund's complete adjuvant. A 1 milliliter inoculum of thismixture was used for the first intradermal injection into a rabbit. Afirst bleed was collected two weeks later. Subsequent injections ofCry3Bb protein designed to boost the immune titer were prepared bymixing equal volumes of 0.2 mg/mL protein with equal volumes of Freund'sincomplete adjuvant. 1 milliliter injections were administered at fourweek intervals, and additional bleeds were obtained every two weeks.Immune serum adequate for analytical purposes was prepared from rabbit#783 after purification over a Protein A Sepharose CL-4B affinitychromatography according to the manufacturers' instructions (SigmaChemical Co, St. Louis, Mo.) and concentrated to 1 milligram of IgGprotein per milliliter and stored in the dark at 4° C. A sample of thisantiserum was conjugated to alkaline phosphatase enzyme for subsequentuse in quantitative ELISA assays.

Leaf and root samples were collected from plants expressing Cry3Bbvariant proteins 11231, 11084, 11098, and 11247. Extracts of plantsamples were prepared as follows. Plant tissue, root or leaf parts, washarvested and weighed on a gram scale. Leaf tissue was mixed with 20parts TBA buffer, weight to volume. Root tissue was mixed with 10 partsTBA buffer, weight to volume. Tissues were ground into an emulsion usinga Wbeaton™ overhead grinder and stored on ice or at −20° C. 250microliters of rabbit anti-Cry3Bb antiserum diluted 1:1000 in carbonatecoating buffer, pH9.6, was distributed onto each well of a 96-wellmicrotiter plate and incubated overnight at 4° C. The plate was thenwashed with PBST (3×5 min). Tissue extract samples were loaded induplicate at 20 microliters per well and at varying dilutions in orderto obtain a value within a standard curve established using Cry3Bbvariant 11231. Plates were incubated overnight at 4° C., then washedwith PBST three times, five minutes each time. 50 microliters of therabbit anti-Cry3B alkaline phosphatase conjugated polyclonal antibodywas added to each well, followed by the addition of 180 uL of PBSTcontaining 1% PVP-40 (Sigma). After overnight incubation, plates werewashed with PBST (3×5 min) and developed with alkaline phosphatase colordevelopment solution consisting of 20 mg para-nitrophenyl phosphate in25 mL diethanolamine, pH9.8, 200 uL/well). Plates were read at λ405after 15-20 minutes, using a quadratic curve fit to a protein standardcurve where the optical density of the highest standard wasapproximately 1.00.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Isolation, Characterization, and Identification of Cry3Proteins and Genes, and Construction of Amino Acid Sequence VariantsThereof

Means for identifying and characterizing Coleopteran toxic gene productsare well documented in the art, and methods for isolating,characterizing and identifying the genes which encode such gene productsare also well known in the art. In addition, the means for producingamino acid sequence variants of such Coleopteran toxic δ-endotoxinproteins are also well known. In particular, Van Rie et al. (U.S. Pat.No. 5,659,123; 1997) identify Cry3A and D toxins which exhibitColeopteran inhibitory properties, and also set forth a method foridentifying mutants which can be constructed which have reducedinsecticidal activity with reference to the wild type protein. Van Rieet al. describe how those particular mutants can be further manipulatedto identify amino acid sequence variant toxins which exhibit increasedinsecticidal activity with reference to the wild type protein. Englishet al. (WO 99/31248) describe other methods and compositions, inparticular for Cry3B, which enable the identification of Cry3 encodinggenes and gene products and the methods which can be used to constructand identify amino acid sequence variants exhibiting improvedinsecticidal activity with reference to that of the wild type Cry3protein. Several coding sequences used herein were derived from thosedescribed in English et al. and the proteins produced from these codingsequences represent in particular the variants 11231 or 11098 asdescribed therein.

Example 2 Construction of Monocot Plant Expression Vectors for theCry3Bb Variants

Design of Cry3Bb Variant Genes for Plant Expression:

For efficient expression of the Cry3Bb variants in transgenic plants,the gene encoding the variants must have a suitable sequence composition(Diehn et al, 1996). One example of such a sequence is shown for thev11231 gene (SEQID NO:7) which encodes the 11231 variant of the Cry3Bbprotein (SEQID NO: 8) exhibiting Diabroticus activity. This gene wasderived via mutagenesis (Kunkel, 1985) of a Cry3Bb synthetic gene (SEQIDNO:5) encoding a protein essentially homologous to the protein encodedby the native Cry3Bb gene (Gen Bank Accession Number m89794; SEQIDNO:1). The following oligonucleotides were used in the mutagenesis ofthe original Cry3Bb synthetic gene (SEQID NO:5) to create the v11231gene (SEQID NO:7)

Oligo #1: TAGGCCTCCATCCATGGCAAACCCTAACAATC (SEQID NO: 40)

Oligo #2: TCCCATCTTCCTACTTACGACCCTGCAGAAATACGGTCCAAC (SEQID NO:41)

Oligo #3: GACCTCACCTACCAAACATTCGATCTTG (SEQID NO: 42)

Oligo #4: CGAGTTCTACCGTAGGCAGCTCAAG (SEQID NO:43)

Construction of Cry3Bb Monocot Plant Expression Vector:

To place the Cry3Bb variant gene v11231 in a vector suitable forexpression in monocotyledonous plants (i.e. under control of theenhanced Cauliflower Mosaic Virus 35S promoter and linker to the hsp70intron followed by a nopaline synthase polyadenylation site as in Brownand Santino U.S. Pat. No. 5,424,412; 1995), the vector pMON19469 wasdigested with NcoI and EcoRI. The larger vector band of approximately4.6 kb was isolated after electrophoresis of the digestion productsthrough an agarose gel, purified, and ligated with T4 DNA ligase to theNcoI-EcoRI fragment of approximately 2 kb containing the v11231 gene(SEQID NO:7). The ligation mix was transformed into a useful laboratorystrain of E. coli, and carbenicillin resistant colonies were recovered.Plasmid DNA was recovered by miniprep DNA procedures from subsequentovernight cultures of carbenicillin resistant colonies selected intobroth containing antibiotics. This DNA was subjected to restrictionendonuclease analysis with enzymes such as NcoI and EcoRI, NotI, andPstI to identify clones containing the v11231 coding sequence fused tothe hsp70 intron under control of the enhanced CaMV35S promoter. Clonesidentified as such were designated as pMON33708.

To place the v11231 gene in a vector suitable for recovery of stablytransformed and insect resistant plants, the 3.75 kb NotI restrictionfragment from pMON33708 containing the lysine oxidase coding sequencefused to the hsp70 intron under control of the enhanced CaMV35S promoterwas isolated and purified after extraction from an agarose gel. Thisfragment was ligated with pMON30460 treated with NotI and calfintestinal alkaline phosphatase. pMON30460 contains the neomycinphosphotransferase coding sequence under control of the CaMV35Spromoter. Kanamycin resistant colonies were obtained by transformationof this ligation mix into E. coli and colonies containing theappropriate band were identified by restriction endonuclease digestionand designated as pMON33710. Restriction enzymes such as NotI, EcoRV,HindIII, NcoI, EcoRI, and BglII were used to identify the appropriateclones containing the NotI fragment of pMON33708 in the NotI site ofpMON30460 (i.e. pMON33710) in the orientation such that both genes arein tandem (i.e. the 3′ end of the v11231expression cassette is linked tothe 5′ end of the nptII expression cassette). Expression of the v11231protein by pMON33710 in corn protoplasts was confirmed byelectroporation of pMON33710 covalently closed circular plasmid DNA intoprotoplasts followed by protein blot and ELISA analysis. This vector canbe introduced into the genomic DNA of corn embryos by particle gunbombardment followed by paromomycin selection to obtain corn plantsexpressing the v11231 gene essentially as described in Brown and SantinoU.S. Pat. No. 5,424,412. In this example, the vector was introduced intoimmature embryo scutella (IES) of maize via co-bombardment along withwith a plasmid conferring hygromycin resistance, followed by hygromycinselection, and regeneration. Transgenic corn lines expressing the v11231protein were identified by ELISA analysis scoring for both the presenceand amount of v11231 protein present in each extract sample. Plants wereselfed and allowed to go to seed. Progeny seed were cured and planted toproduce seedling corn plants which were subsequently tested forprotection from Diabroticus feeding.

In Plant Performance of Cry3Bb Variant 11231:

Transformed corn plants expressing Cry3Bb variant 11231 protein werechallenged with western corn rootworm (WCR) larvae in both a seedlingand 10 inch pot assay. The transformed genotype was A634, where theprogeny of the R0 cross by A634 was evaluated. Observations includedeffect on larval development (weight), root damage rating (RDR), andprotein expression. The transformation vector containing the Cry3Bbvariant gene was pMON33710. Treatments included the positive andnegative iso-populations for each event and an A634 check.

The seedling assay consisted of the following steps; i. single seedswere placed in 1 oz cups containing potting soil; ii. at spiking, eachseedling was infested with 4 neonate larvae, and iii. after infestation,seedlings were incubated for 7 days at 25° C., 50% RH, and 14:10 (L:D)photo period. Adequate moisture was added to the potting soil during theincubation period to maintain seedling vigor.

The 10 inch pot assay consisted of the following steps; i. single seedswere placed in 10 inch pots containing potting soil; ii. at 14 days postplanting, each pot was infested with 800 eggs which have beenpre-incubated such that hatch would occur 5-7 days post infestation; andiii. after infestation, plants were incubated for 4 weeks under the sameenvironmental conditions as the seedling assay. Pots were both sub & topirrigated daily.

For the seedling assay, on day 7 plants were given a root damage rating(Table 1.) and surviving larvae were weighed. Also at this time, Cry3Bbprotein concentrations in the roots were determined by ELISA.

Table 1. Root Damage Rating Scale for Seedling Assay.

RDR 0=no visible feeding

1=very light feeding

2=light feeding

3=moderate feeding

4=heavy feeding

5=very heavy feeding

Results of the seedling assay are shown in Table 2. Plants expressingCry3Bb protein were completely protected by WCR feeding, where survivinglarvae within this treatment had not grown. Mean larval weights rangedfrom 2.03-2.73 mg for the non-expressing treatments, where the survivinglarval average weight was 0.11 mg on the expressing Cry3Bb treatment.Root damage ratings were 3.86 and 0.33 for the non-expressing andexpressing iso-populations, respectively. Larval survival ranged from75-85% for the negative and check treatments, where only 25% of thelarvae survived on the Cry3Bb treatment.

TABLE 2 Effect of Cry3Bb expressing plants on WCR larvae in a seedlingassay. Plants Larvae Root % Mean ± SD Event Treatment N (ppm) RDR ± SD NSurv Wt. (mg) 16 Negative 7 0.0 3.86 ± 0.65 21 75 2.73 ± 1.67 16Positive 3 29.01 0.33 ± 0.45  3 25 0.11 ± 0.07 A634 Check 4 0.0 — 13 812.03 ± 0.83

For the 10 inch pot assay, at 4 weeks post infestation plant height wasrecorded and a root damage rating was given (Iowa 1-6 scale; Hills, T.M. and D. C. Peters. 1971; A method of evaluating post plantinginsecticide treatments for control of western corn rootworm larvae.Journal of Economic Entomology 64: 764-765.).

Results of the 10 inch pot assay are shown in Table 3. Plants expressingCry3Bb protein had significantly less feeding damage and were tallerthan the non-expressing plants. Event 16, the higher of the twoexpressing events provided nearly complete control. The negativetreatments had very high root damage ratings indicating very high insectpressure. The positive mean root damage ratings were 3.4 and 2.2 forevent 6 & 16, respectively. Mean RDR for the negative treatment was 5.0& 5.6.

TABLE 3 Effect of Cry3Bb expressed in corn in controlling WCR larvalfeeding in a 10 inch pot assay. Root Plant Event Treatment N (ppm) RDR ±SD Height (cm)  6 Negative 7 0.0 5.0 ± 1.41 49.7 ± 18.72  6 Positive 57.0 3.4 ± 1.14 73.9 ± 8.67  16 Negative 5 0.0 5.6 ± 0.89 61.2 ± 7.75  16Positive 5 55.0 2.2 ± 0.84 83.8 ± 27.15

In summary, corn plants expressing Cry3Bb protein have a significantbiological effect on WCR larval development as seen in the seedlingassay. When challenged with very high infestation levels, plantsexpressing the Cry3Bb protein were protected from WCR larval feedingdamage as illustrated in the 10 inch pot assay.

Example 3 Increased Expression of a Cry3Bb Protein in Transgenic Maize

Expression of a Cry3Bb protein was compared in corn plants transformedwith standard or preferred Cry3Bb expression vectors. Plants transformedwith the improved vectors consistently demonstrated significantly higherlevels of expression of Cry3Bb when compared to plants transformed withthe standard Cry3Bb vectors. A standard Cry3Bb plant expression vectorpMON33710 contains an expression cassette composed of an enhancedCaMV35S promoter sequence (P-CaMV.35S, SEQID NO:29), a Zea mays Hsp70intron sequence (I-Zm.Hsp70, SEQID NO:33), a non-naturally occurringsequence encoding Cry3Bb variant protein v11231 (Bt.cry3Bb.v11231, SEQIDNO: 7), and a nopaline synthase transcription termination andpolyadenylation sequence (T-AGRtu.nos, SEQID NO:34). Another standardCry3Bb plant expression vector pMON33709 contains an expression cassettecomposed of an enhanced CaMV35S promoter sequence (P-CaMV.35S, SEQIDNO:29), a Zea mays Hsp70 intron sequence (I-Zm.Hsp70, SEQID NO:33), aZea mays CTP encoding sequence (TS-Zm.rbc1, SEQID NO:25), anon-naturally occurring sequence encoding Cry3Bb variant protein v11231(Bt.cry3Bb.v11231, SEQID NO:7), and a nopaline synthase transcriptiontermination and polyadenylation sequence (T-AGRtu.nos, SEQID NO:34). Theplant expression vector pMON25097 is improved compared to pMON33710 asjudged by Cry3Bb expression levels in planta, and contains an expressioncassette comprising a non-naturally occurring CaMV35S AS4 promotersequence (P-CaMV.AS4, SEQID NO:30), a wheat chlorophyll A/B bindingprotein untranslated leader sequence (L-Ta.hcb1, SEQID NO:31), a riceactin intron sequence (I-Os.Act1, SEQID NO:32), and a non-naturallyoccurring sequence encoding Cry3Bb variant protein 11231mv1 (11098)(Bt.cry3Bb.11231mv1, SEQID NO:9) linked to a wheat heat shock Hsp17transcription termination and polyadenylation sequence (T-Ta.Hsp17,SEQID NO:35). Another preferred vector is pMON25096, which contains anexpression cassette (SEQID NO:17) comprising a non-naturally occurringCaMV35S AS4 promoter sequence (P-CaMV.AS4, SEQID NO:30), a wheatchlorophyll A/B binding protein untranslated leader sequence (L-Ta.hcb1,SEQID NO:31), a rice actin intron sequence (I-Os.Act1, SEQID NO:32), aZea mays CTP encoding sequence (TS-Zm.rbc1, SEQID NO:25), and anon-naturally occurring sequence encoding Cry3Bb variant protein11231mv1 (Bt.cry3Bb.11231mv1, SEQID NO:9) linked to a wheat heat shockHsp17 transcription termination and polyadenylation sequence(T-Ta.Hsp17, SEQID NO:35). All vectors contain an identical cassettelinked to the Cry3Bb expression cassette which confers paromomycinresistance to transformed plant tissue. This resistance cassetteconsists of an enhanced CaMV35S promoter sequence, and a neomycinphosphotransferase coding sequence linked to a nopaline synthasetranscription termination and polyadenylation sequence. A summary of thestandard and improved vectors is presented in Table 4. Transgenic cornplants resistant to paromomycin were derived essentially as described inU.S. Pat. No. 5,424,412 (1995).

TABLE 4 Plant Expression Vector Summary Selection Vector ExpressionCassette Cassette pMON33709 35S/HSP70/ZmRBC/v11231/NOS e35S/nptII/nospMON33710 e35S/HSP70/11231v/nos e35S/nptII/nos pMON33722AS4/TaCAB/OsAct1/ZmRBC/v11231/ e35S/nptII/nos tahsp17 pMON33723AS4//TaCAB/OsAct1/ v11231/tahsp17 e35S/nptII/nos pMON25096AS4/TaCAB/OsAct1/ZmRBC/11231mv1/ e35S/nptII/nos tahsp17 pMON25097AS4/TaCAB/OsAct1/11231mv1/tahsp17 e35S/nptII/nos pMON33741AS4/TaCAB/OsAct1/11231mv2/tahsp17 e35S/nptII/nos pMON33748e35S/TaCAB/OsAct1/11231mv2/tahsp17 e35S/nptII/nos

Maize leaf protoplasts were electroporated with standard vectors(pMON33709 or pMON33710) or improved vectors (pMON33722, pMON33723,pMON25096, pMON25097, pMON33741) as described (Sheen, Plant Cell2:1027-1038, 1990) and transient expression of Cry3Bb variant proteinswas compared by ELISA and Western Blot analysis methods. The ELISA useda rabbit anti-Cry3B chromatography purified IgG capture antibody raisedagainst Cry3B 11231, a sample of that antibody conjugated to alkalinephosphatase as the secondary detecting antibody, and a purified Cry3Bbnative protein as a standard. Comparison of the ratio of Cry3Bb toneomycin phosphotransferase (Npt II) expression levels by ELISAindicated that approximately two-fold increases in the normalizedexpression levels of Cry3Bb variant protein 11231 were obtained withimproved vectors pMON33723 and pMON33722 relative to the standardvectors pMON33710 and pMON33709, respectively.(Expt. 1, Table 5).,Differences in Cry3Bb expression are directly ascribed to the improvedexpression cassette in the improved vectors rather than to differencesin protoplast electroporation efficiency since expression of Cry3Bbprotein is normalized to Npt II produced by the identical linked nptIIgene present in all vectors. The most preferred improved vectors such aspMON25096, pMON25097, and pMON33741 expressed approximately 10-foldhigher normalized levels of Cry3Bb and variant Cry3Bb protein than thepreferred improved vectors such as pMON33722 or pMON33723 (Table 5,Expt. 2, 3). Finally, the equally preferred pMON33741 and pMON25097vectors yielded roughly equivalent normalized Cry3Bb expression (Table5, Expt. 4).

TABLE 5 Transient Cry3Bb and Cry3Bb Variant Expression in Corn LeafProtoplasts (normalized to NptII expression) Expt. 1 pMON33710 pMON337235.79 12.3 pMON33709 pMON33722 2.7 7.7 Expt. 2 pMON33722 pMON25096 1.926.2 pMON33723 pMON25097 3.7 37.5 Expt. 3 pMON33723 pMON33741 30 319Expt. 4 pMON33741 pMON25097 20 25

Since the improved expression cassette in pMON25097 encodes the Cry3Bb11231mv1 (11098) variant toxin, and the standard cassette in pMON33710encodes the Cry3Bb v11231 variant which differ by a single amino acid,the intrinsic immunoreactivity of the two proteins in the ELISA assaywas compared. Subsequent ELISA experiments with Cry3Bb v11231 and11231mv1 (11098) variant proteins produced in and purified from B.thuringiensis indicate that the two proteins have similar levels ofimmunoreactivity. Consequently, the observed increase in levels ofCry3Bb 11231mv1 (11098) protein produced from the expression cassette inpMON25097 is due to increased expression levels rather than a differencein immunoreactivity. Protein blot analyses confirm that the increasedlevel of cross reactive material produced in maize protoplasts from theimproved Cry3Bb expression cassette in pMON25097 were due to increasedaccumulation of an approximately 60,000 Mr protein immunoreactive withCry3B antiserum that also co-migrates with Cry3Bb variant 11231 proteinproduced in a recombinant cry-B. thuringiensis strain from pEG7174.Equally preferred and improved Cry3Bb variant protein expressioncassettes in pMON33741 and pMON33748 that encode Cry3Bb.11231 alsoexhibit increased expression levels of Cry3Bb relative to expressionobserved from the standard cassette in pMON33710. These results confirmthat expression differences are due to the improved compositionsdisclosed herein rather than to differences in the intrinsicimmunoreactivity of the different variants.

Root tissue from transgenic plants in the R₀ stage independentlyobtained after transformation with an improved vectors (pMON33723,25097,) or with a standard vector (pMON 33710) was subjected toquantitative analysis of Cry3Bb protein levels by a quantitative ELISAassay. Comparison of Cry3Bb or Cry3Bb protein variant expression levelsin improved and standard vector transformed corn plants show thatCry3Bb.11231 variant expression does not exceed 50 ppm in the standardpMON33710 transgenics while Cry3Bb.11098 (11231mv1) expression in theimproved pMON25097 transgenics is frequently higher than 50 ppm (Table6). Protein blot analyses confirm that the increased level of crossreactive material produced by pMON25097 (improved) were due to increasedaccumulation of an approximately Mr 60,000 protein that migrates withCry3Bb1 standard from B. thuringiensis. Other improved Cry3Bb proteinvariant expression cassettes found in pMON33741 and 33748 alsoconsistently yield select independently transformed events (ITE's) withCry3Bb protein variant levels greater than 100 PPM whereas the standardvectors have never given rise to IFE's with greater than 50 PPM ofCry3Bb protein variant (Table 7). High level expression is evident inboth the H99 and A634 maize genotypes, indicating that the compositionsdisclosed herein have broad utility to many varieties of commerciallycultivated maize. Such select high expressing Cry3 protein variant linesobtained with the vectors described herein are expected to be especiallyadvantageous in conferring high levels of protection to insect feedingdamage and in reducing the incidence of insect resistance to Cry3insecticidal proteins.

TABLE 6 Comparison of Cry3Bb Expression in R₀ Corn Transformed withStandard and Improved Cry3Bb Protein Variant Expression Cassettes Cry3BExpression Level (ppm) Vector Total 5-10 10-50 50-100 100-200 >200(genotype) Events ppm ppm ppm ppm ppm L25097 A634 45 3 7 3 H99 589 32 365 3 5 L33710 A634 22 2 2 H99 336 13 15 L33723 A634 0 H99 67 6 9

TABLE 7 Cry3Bb Expression in R₀ Corn Transformed with Improved Cry3BbProtein Variant Expression Cassettes Cry3B Expression Level (ppm) Total5-10 10-50 50-100 100-200 >200 Vector Events ppm ppm ppm ppm ppm L25097A634 112 7 4 5 1 4 H99 45 1 4 2 L33741 H99 108 11 5 2 4 L33748 A634 82 111 2 2 1 H99 209 23 13 3 3 11

Progeny derived from corn plants transformed with both the standard(pMON33709 and pMON33710) and preferred (pMON25096, 25097, 33722, 33723,33726, 33741, and 33748) cassettes expressing 10 ppm or more of Cry3Bbprotein were further tested for resistance to Corn Rootworm (CRW)feeding damage in greenhouse or growth chamber based bioassays aspreviously described (English et al., WO 99/31248). Corn Rootwormresistant transgenic corn plants were obtained from essentially all ofthe preferred vectors (Table 8). For example, the improved pMON25096vector was used to generate 89 independently transformed events (ITE's),14 independent pMON25096 F₁ progeny lines expressing 10 ppm or more ofCry3Bb and 7 F₁ progeny lines displaying significant levels of CRWresistance (an RDR rating≧3.5 on a rating scale of 0-6). In contrast,not a single event with a RDR rating≦3.5 was obtained from 12 of thestandard pMON33710 cassette F₁ progeny lines expressing 10 PPM or moreof Cry3Bb protein variant. Failure to obtain CRW resistant lines witheither of the standard vectors (pMON33709 or pMON33710) was not due toinsufficient numbers of ITE's as over 300 ITE's from each of these twovectors were generated and screened for CRW resistant F₁ progeny. Farfewer ITE's were generated with preferred vectors such as pMON33722,pMON33723, and pMON25096, yet all ultimately gave rise to CRW resistantF₁ progeny lines.

TABLE 8 Numbers of CRW resistant independent transformation eventsobtained with the standard and improved Cry3Bb Protein Variantexpression cassettes Number Number and Expression Total Number of ofITE's Percent of ITEs cassette Genotype ITE's Tested with RDR < 3.5L33709 H99 318 11 0 L33710 H99 336 10 0 A634 22 2 0 L25096 H99 52 4 2(50%) A634 37 10 5 (50%) L25097 H99 634 17 10 (59%)  A634 157 18 8 (44%)L33722 H99 107 10 6 (60%) L33723 H99 93 7 3 (43%) L33726 H99 65 6 5(83%) A634 10 0 L33727 H99 86 0 A634 1 1 0 33736ABI H99 3 3 2 (67%)L33741 H99 108 1 0 L33748 H99 223 6 3 (50%) A634 82 7 4 (57%) L33749ABIH99 73 14 13 (93%) 

In examples provided herein, experimental evidence that substantiallyequivalent compositions based on the improvements disclosed herein yieldequivalent improvements in performance relative to the previouslydisclosed standards. More specifically, we demonstrate that improvedcompositions encoding both the Cry3Bb.11098 and Cry3Bb.11231 variantsboth yield equivalently improved performance relative to the previouslydisclosed standard compositions encoding Cry3Bb.11231. It thus followsthat use of other Cry3B variants with specific biological activitiesthat are greater than or equal to Cry3Bb.11098 or Cry3Bb.11231 iscontemplated by and within the scope of this invention. For example,improved vector compositions encoding Cry3Bb variants include 11231,11084, 11098, 11247, and others as set forth in English et al., U.S.application Ser. Nos. 08/993,170, 08/993,722, 08/993,755, and08/996,441, all filed Dec. 18, 1997 can be derived from pMON25095 usingstandard mutagenesis procedures in a manner essentially equivalent tothe construction of pMON33740.

Example 4 Preferred Expression Cassettes Confer Resistance to CRW Damagein Field Tests

Corn plants genetically modified to express Cry3Bb protein variantsderived from the preferred vectors pMON33722, pMON33723, pMON25096, andpMON25097 were evaluated in the field for control of western cornrootworm, Diabrotica vergifera vergifera LeConte (WCR). None of the cornplants transformed with the standard vectors were advanced to fieldtesting as none displayed adequate Corn Rootworm control in greenhousetests (Example 3. Table 8). The efficacy trials were held at a Monsantoresearch farm in Jerseyville, Ill. and at the Northern Grain InsectsResearch Laboratory, USDA ARS research station in Brookings, SouthDakota. These trials serve to evaluate performance of the preferredcassettes in the field under heavy insect pressure and to compare theirperformance to the current commercially available insecticides.

Seventeen independent transformation events (ITE) were selected forfield evaluation based on greenhouse performance. The amount of seedavailable for the field evaluation varied for each ITE. Of these 17events, only seven were planted at the Brookings research station. Thefield design for the Brookings' location was a randomized complete block(RCB) with 2 replications, where each plot was a single row containing amaximum of 30 plants. All 17 ITE's were planted at the Jerseyvillelocation, where the design was a RCB with a maximum of 4 replications, 1row plots each, where the number of replications depended on the seedavailable from each ITE. Because of this, the number of replications atJerseyville ranged from two to four. Additional treatments included anuntreated check (nontransgenic corn) and commercial insecticides,including Counter®, Lorsban®, and Force®. The insecticide treatmentswere only at the Jerseyville location. The insecticides were applied asan eight inch band at planting using the recommended rates.

Planting dates where May 28^(th) and June 3^(rd) for the Jerseyville &Brookings, respectively. The study was performed as follows; plots wereinfested with CRW eggs at planting with 1,600 eggs per foot of row,approximately 800 eggs per plant. At the V1-V2 plant growth stage,plants were analyzed for presence of the Cry3Bb protein variantexpression using an ELISA. Plants negative for the gene were culled fromthe plot.

At the end of the CRW larval feeding stage, when maximum damage wouldhave occurred, all remaining plants in each plot were evaluated for rootfeeding injury using a 1-6 root damage rating (RDR) scale described byHills and Peters (1971). The RDR scale is as follows;

Root Damage Rating:

1. No feeding scars

2. Visible feeding scars, but no roots pruned to within 4 cm of thestalk

3. One or more nodal roots pruned to within 4 cm of the stalk, but lessthan one nodes worth of roots

4. One node worth of pruned roots

5. Two nodes worth of pruned roots

6. Three or more nodes worth of pruned roots

On July 25^(th) and August 3^(rd) the field trials were evaluated atJerseyville and Brookings, respectively. The average RDR's for alltreatments are illustrated in Table 9. Of the seventeen ITE's evaluated,16 ITE's controlled CRW feeding, ≦3.0 RDR. Two of the three chemicalstandards had a RDR less than 3.0. Force® had a root damage rating of3.2. Except for one ITE, WCR20, all treatments were significantly betterthan the checks (p<0.01) but did not differ significantly from eachother. Figure one illustrates the difference in larval feeding damagebetween a transgenic CRW resistant plant and an untreated check.

Even though the ITE's did not differ significantly from the chemicalstandards with respect to root damage rating, the amount of feedinginjury observed on roots from the insecticide treatments were greaterthan the roots expressing Monsanto's proprietary gene. The lack ofdifference between root damage rating is an artifact of the root ratingscale, where this scale is based on “pruned” roots. Hills and Petersdescribe a pruned root as being less than 4 cm in length due to CRWfeeding. Therefore, root masses without a “pruned” root but visiblefeeding scares are given a rating of 2. Roots outside of the zone ofprotection from the insecticide treatments had many more feeding scarsand in most cases the root tips were destroyed as compared to the ITE's.Unlike the insecticide treatments, the transgenic plants express the CRWresistant gene throughout the entire root mass. But because themechanism for control of the transgenic plant is orally mediated, aminimum amount of feeding is required to control any further injury bythe CRW larvae. This minimal feeding requirement resulted in a RDR of 2.

In summary, corn plants expressing Cry3Bb protein variants were fullyprotected from CRW larval feeding. This level of protection eliminatesthe need for an insecticide treatment. Insecticides, includingorganophosphates, carbamates and pyrethroids are incorporated into thesoil on over 16 million corn acres annually to control CRW. CRWresistance technology has the potential to significantly reduce thecurrent exposure level of these insecticides to the environment. Thebenefits of shifting away from soil insecticides to a transgenicapproach are impressive and include a reduction in potential humanhealth and safety risks, reduced direct impacts on nontarget organisms,reduced contamination of surface and ground water supplies, decreasedpesticide container disposal problems, and general compatibility withother pest management and agronomic programs.

TABLE 9 Corn rootworm root feeding damage (RDR) means for cornindependent transformation events containing Monsanto's proprietary CRWresistant gene. Root Damage Rating (RDR) Treatment Jerseyville BrookingsAverage (RDR) pMON 25097-1 2.3 1.9 2.1 pMON 33722-1 2.6 2.3 2.5 pMON33723-1 2.6 2.9 2.8 pMON 33723-2 2.6 2.0 2.3 pMON 33722-2 2.5 1.9 2.2pMON 25096-1 2.8 2.5 2.7 pMON 25097-2 2.5 2.3 2.4 pMON 25096-2 2.4 n/a2.4 pMON 25097-3 2.6 n/a 2.6 pMON 25096-3 2.2 n/a 2.2 pMON 25097-4 2.2n/a 2.2 pMON 25096-4 2.6 n/a 2.6 pMON 33723-3 2.5 n/a 2.5 pMON 25097-53.0 n/a 3.0 pMON 25097-6 4.0 n/a 4.0 pMON 25097-7 2.2 n/a 2.2 pMON33722-3 2.6 n/a 2.6 COUNTER ® 2.4 n/a 2.4 LORSBAN ® 2.4 n/a 2.4 FORCE ®3.2 n/a 3.2 CHECK 4.1 4.1 4.1

Example 5 Transformation of Tobacco Chloroplast with a Cry3B Gene

Recombinant plants can be produced in which only the mitochondrial orchloroplast DNA has been altered to incorporate the molecules envisionedin this application. Promoters which function in chloroplasts have beenknown in the art (Hanley-Bowden et al., Trends in Biochemical Sciences12:67-70, 1987). Methods and compositions for obtaining cells containingchloroplasts into which heterologous DNA has been inserted have beendescribed, for example by Daniell et al. (U.S. Pat. No. 5,693,507; 1997)and Maliga et al. (U.S. Pat. No. 5,451,513; 1995). A vector can beconstructed which contains an expression cassette from which a Cry3Bprotein could be produced. A cassette could contain a chloroplastoperable promoter sequence driving expression of a cry3B crystal proteingene, constructed in much the same manner as other polynucleotidesherein, using thermal amplification methodologies, restrictionendonuclease digestion, and ligation etc. A chloroplast expressible genewould provide a promoter and a 5′ untranslated region from aheterologous gene or chloroplast gene such as psbA, which would providefor transcription and translation of a DNA sequence encoding a Cry3Bprotein in the chloroplast; a DNA sequence encoding Cry3B protein; and atranscriptional and translational termination region such as a 3′inverted repeat region of a chloroplast gene that could stabilize anexpressed cry3B mRNA. Expression from within the chloroplast wouldenhance cry3B gene product accumulation. A host cell containingchloroplasts or plastids can be transformed with the expression cassetteand then the resulting cell containing the transformed chloroplasts canbe grown to express the Cry3B protein. A cassette may also include anantibiotic, herbicide tolerance, or other selectable marker gene inaddition to the cry3B gene. The expression cassette may be flanked byDNA sequences obtained from a chloroplast DNA which would facilitatestable integration of the expression cassette into the chloroplastgenome, particularly by homologous recombination. Alternatively, theexpression cassette may not integrate, but by including an origin ofreplication obtained from a chloroplast DNA, would be capable ofproviding for replication of the heterologous cry3B gene in thechloroplast. Plants can be generated from cells containing transformedchloroplasts and can then be grown to produce seeds, from whichadditional plants can be generated. Such transformation methods areadvantageous over nuclear genome transformation, in particular wherechloroplast transformation is effected by integration into thechloroplast genome, because chloroplast genes in general are maternallyinherited. This provides environmentally “safer” transgenic plants,virtually eliminating the possibility of escapes into the environment.Furthermore, chloroplasts can be transformed multiple times to producefunctional chloroplast genomes which express multiple desiredrecombinant proteins, whereas nuclear genomic transformation has beenshown to be rather limited when multiple genes are desired.Segregational events are thus avoided using chloroplast or plastidtransformation. Unlike plant nuclear genome expression, expression inchloroplasts or plastids can be initiated from only one promoter andcontinue through a polycistronic region to produce multiple peptidesfrom a single mRNA.

The expression cassette would be produced in much the same way thatother plant transformation vectors are constructed. Plant chloroplastoperable DNA sequences can be inserted into a bacterial plasmid andlinked to DNA sequences expressing desired gene products, such as Cry3Bproteins, so that Cry3B protein is produced within the chloroplast,obviating the requirement for nuclear gene regulation, capping,splicing, or polyadenylation of nuclear regulated genes, or chloroplastor plastid targeting sequences. An expression cassette comprising acry3B gene, which is either synthetically constructed or a native genederived directly from a B. thuringiensis genome or a B. thuringiensisepisomal element, would be inserted into a restriction site in a vectorconstructed for the purpose of chloroplast or plastid transformation.The cassette would be flanked upstream by a chloroplast or plastidfunctional promoter and downstream by a chloroplast or plastidfunctional transcription and translation termination sequence. Theresulting cassette would be incorporated into the chloroplast or plastidgenome using well known homologous recombination methods.

Alternatively, chloroplast or plastid transformation could be obtainedby using an autonomously replicating plasmid or other vector capable ofpropagation within the chloroplast or plastid. One means of effectuatingthis method would be to utilize a portion of the chloroplast or plastidgenome required for chloroplast or plastid replication initiation as ameans for maintaining the plasmid or vector in the transformedchloroplast or plastid. A sequence enabling stable replication of achloroplast or plastid epigenetic element would easily be identifiedfrom random cloning of a chloroplast or plastid genome into a standardbacterial vector which also contains a chloroplast or plastid selectablemarker gene, followed by transformation of chloroplasts or plastids andselection for transformed cells on an appropriate selection medium.Introduction of an expression cassette as described herein into achloroplast or plastid replicable epigenetic element would thus providean effective means for localizing a Cry3B B. thuringiensis δ-endotoxinto the chloroplast or plastid.

Example 6 Targeting Cry3Bb or Variant Cry3Bb Protein to Plastids

Improved expression by targeting recombinant insecticidal protein to thechloroplast may result in tissues which are light exposed and whichaccumulate mature chloroplasts as a result. Improving expression in leaftissue to inhibit leaf-feeding pests susceptible to the insecticidalprotein could be advantageous. To test this, two plasmids, pMON33709 andpMON33710 were constructed which were isogenic with respect to allelements with the exception of a plastid or chloroplast targetingsequence linked in frame to the insecticidal Cry3Bb improved variant inpMON33709. R₀ corn plants were recovered and were shown to contain andexpress the transgene by ELISA. Six pMON33709 lines and sixteenpMON33710 lines were recovered which expressed the transgene in both theroot and the leaves. Leaf and root tissue were recovered and analyzedfor the presence and amount of Cry3Bb variant protein, measured in partsper million. The results are shown in Table 10.

TABLE 10 Comparison of Non-Targeted and Plastid Targeted Leaf vs RootExpression of Cry3Bb Variant v11231 in R₀ Corn Transformation Events R0# Event # Construct Tissue ppm 11231 ug/g tissue R053608 2027-05-01L33709 Leaf 14.69 R053608 2027-05-01 L33709 Root 3.97 R053621 2028-06-06L33709 Leaf 22.65 R053621 2028-06-06 L33709 Root 0.10 R053643 2029-03-09L33709 Leaf 1.05 R053643 2029-03-09 L33709 Root 3.83 R053675 2028-03-06L33709 Leaf 7.13 R053675 2028-03-06 L33709 Root 2.23 R053688 2028-04-02L33709 Leaf 56.80 R053688 2028-04-02 L33709 Root 9.83 R053690 2028-04-02L33709 Leaf 98.69 R053690 2028-04-02 L33709 Root 6.38 R053708 2027-01-02L33710 Leaf 12.79 R053708 2027-01-02 L33710 Root 4.94 R053781 2028-02-19L33710 Leaf 8.47 R053781 2028-02-19 L33710 Root 4.72 R053785 2027-04-06L33710 Leaf 21.97 R053785 2027-04-06 L33710 Root 7.20 R053799 2028-01-16L33710 Leaf 12.41 R053799 2028-01-16 L33710 Root 6.19 R053800 2028-01-16L33710 Leaf 5.69 R053800 2028-01-16 L33710 Root 3.32 R053801 2028-01-16L33710 Leaf 16.19 R053801 2028-01-16 L33710 Root 7.80 R053824 2027-01-11L33710 Leaf 6.93 R053824 2027-01-11 L33710 Root 10.35 R053838 2030-08-12L33710 Leaf 14.32 R053838 2030-08-12 L33710 Root 5.64 R053857 2030-08-08L33710 Leaf 12.70 R053857 2030-08-08 L33710 Root 3.97 R053858 2028-02-32L33710 Leaf 2.33 R053858 2028-02-32 L33710 Root 4.15 R053859 2028-02-32L33710 Leaf 9.39 R053859 2028-02-32 L33710 Root 5.76 R053904 2027-02-03L33709 Leaf 226.05 R053904 2027-02-03 L33709 Root 1.55 R0539232029-01-08 L33710 Leaf 12.16 R053923 2029-01-08 L33710 Root 11.77R053924 2029-01-08 L33710 Leaf 10.74 R053924 2029-01-08 L33710 Root 7.94R053928 2029-01-05 L33710 Leaf 14.86 R053928 2029-01-05 L33710 Root 3.84R053929 2029-01-05 L33710 Leaf 15.04 R053929 2029-01-05 L33710 Root 3.49

All but one pMON33709 line (Ro53643) produced between 3 to 15 times moreinsecticidal protein in the leaves than in the root tissue. The one linethat produced less in the leaves also produced less than 1 ppm in theroot, whereas the other lines produced up to almost 100 ppm in theleaves. The amount of Cry3Bb variant protein expressed was even morevariable in the non-targeted lines derived from pMON33710 transformationevents which were determined to be expressing the recombinant protein inboth leaf and root tissues. While most of these lines produced moreprotein in the leaves than in the roots, some also produced more in theroots, but the difference between the amount produced in the roots inthose improved root-expressors was less substantial than in the singlepMON33709 targeted event. Also, the range of expression levels was lesspronounced in the non-targeted events with one exception. Surprisingly,one line (Ro53904) produced substantially more protein in the leavesthan was observed in any other line, targeted or non-targeted. This linewould be expected to be a candidate for a commercial line directed toprotection against Coleopteran pests which feed on leaf tissues.Conversely, lines such as Ro53923 would be expected to be optimumcandidates for protecting corn plants against root-feeding pests such ascorn rootworms.

The data in summary indicates that targeting the Bt Cry3B protein to theplastid or chloroplast improves the accumulation of the protein in leaftissue but not in root tissue, and improves the overall expression ofthe protein in leaves in plants transformed with such constructs ascompared to the levels of expression observed in root tissues in thosesame plants.

In view of the above, it will be seen that the several advantages of theinvention are achieved and other advantageous results attained. Asvarious changes could be made in the above methods and compositionswithout departing from the scope of the invention, it is intended thatall matter contained in the above description and shown in theaccompanying drawings shall be interpreted as illustrative and not in alimiting sense.

In addition, all references referred to in this application are hereinincorporated by reference in their entirety.

43 1 1959 DNA Bacillus thuringiensis CDS (1)..(1956) Description ofArtificial Sequence naturally occurring nucleotide sequence encoding aCry3Bb1 amino acid sequence 1 atg aat cca aac aat cga agt gaa cat gatacg ata aag gtt aca cct 48 Met Asn Pro Asn Asn Arg Ser Glu His Asp ThrIle Lys Val Thr Pro 1 5 10 15 aac agt gaa ttg caa act aac cat aat caatat cct tta gct gac aat 96 Asn Ser Glu Leu Gln Thr Asn His Asn Gln TyrPro Leu Ala Asp Asn 20 25 30 cca aat tca aca cta gaa gaa tta aat tat aaagaa ttt tta aga atg 144 Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys GluPhe Leu Arg Met 35 40 45 act gaa gac agt tct acg gaa gtg cta gac aac tctaca gta aaa gat 192 Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser ThrVal Lys Asp 50 55 60 gca gtt ggg aca gga att tct gtt gta ggg cag att ttaggt gtt gta 240 Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu GlyVal Val 65 70 75 80 gga gtt cca ttt gct ggg gca ctc act tca ttt tat caatca ttt ctt 288 Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln SerPhe Leu 85 90 95 aac act ata tgg cca agt gat gct gac cca tgg aag gct tttatg gca 336 Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe MetAla 100 105 110 caa gtt gaa gta ctg ata gat aag aaa ata gag gag tat gctaaa agt 384 Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala LysSer 115 120 125 aaa gct ctt gca gag tta cag ggt ctt caa aat aat ttc gaagat tat 432 Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu AspTyr 130 135 140 gtt aat gcg tta aat tcc tgg aag aaa aca cct tta agt ttgcga agt 480 Val Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu ArgSer 145 150 155 160 aaa aga agc caa gat cga ata agg gaa ctt ttt tct caagca gaa agt 528 Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln AlaGlu Ser 165 170 175 cat ttt cgt aat tcc atg ccg tca ttt gca gtt tcc aaattc gaa gtg 576 His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys PheGlu Val 180 185 190 ctg ttt cta cca aca tat gca caa gct gca aat aca cattta ttg cta 624 Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His LeuLeu Leu 195 200 205 tta aaa gat gct caa gtt ttt gga gaa gaa tgg gga tattct tca gaa 672 Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr SerSer Glu 210 215 220 gat gtt gct gaa ttt tat cat aga caa tta aaa ctt acacaa caa tac 720 Asp Val Ala Glu Phe Tyr His Arg Gln Leu Lys Leu Thr GlnGln Tyr 225 230 235 240 act gac cat tgt gtt aat tgg tat aat gtt gga ttaaat ggt tta aga 768 Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu AsnGly Leu Arg 245 250 255 ggt tca act tat gat gca tgg gtc aaa ttt aac cgtttt cgc aga gaa 816 Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg PheArg Arg Glu 260 265 270 atg act tta act gta tta gat cta att gta ctt ttccca ttt tat gat 864 Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe ProPhe Tyr Asp 275 280 285 att cgg tta tac tca aaa ggg gtt aaa aca gaa ctaaca aga gac att 912 Ile Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu ThrArg Asp Ile 290 295 300 ttt acg gat cca att ttt tca ctt aat act ctt caggag tat gga cca 960 Phe Thr Asp Pro Ile Phe Ser Leu Asn Thr Leu Gln GluTyr Gly Pro 305 310 315 320 act ttt ttg agt ata gaa aac tct att cga aaacct cat tta ttt gat 1008 Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys ProHis Leu Phe Asp 325 330 335 tat tta cag ggg att gaa ttt cat acg cgt cttcaa cct ggt tac ttt 1056 Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu GlnPro Gly Tyr Phe 340 345 350 ggg aaa gat tct ttc aat tat tgg tct ggt aattat gta gaa act aga 1104 Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn TyrVal Glu Thr Arg 355 360 365 cct agt ata gga tct agt aag aca att act tcccca ttt tat gga gat 1152 Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser ProPhe Tyr Gly Asp 370 375 380 aaa tct act gaa cct gta caa aag cta agc tttgat gga caa aaa gtt 1200 Lys Ser Thr Glu Pro Val Gln Lys Leu Ser Phe AspGly Gln Lys Val 385 390 395 400 tat cga act ata gct aat aca gac gta gcggct tgg ccg aat ggt aag 1248 Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala AlaTrp Pro Asn Gly Lys 405 410 415 gta tat tta ggt gtt acg aaa gtt gat tttagt caa tat gat gat caa 1296 Val Tyr Leu Gly Val Thr Lys Val Asp Phe SerGln Tyr Asp Asp Gln 420 425 430 aaa aat gaa act agt aca caa aca tat gattca aaa aga aac aat ggc 1344 Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp SerLys Arg Asn Asn Gly 435 440 445 cat gta agt gca cag gat tct att gac caatta ccg cca gaa aca aca 1392 His Val Ser Ala Gln Asp Ser Ile Asp Gln LeuPro Pro Glu Thr Thr 450 455 460 gat gaa cca ctt gaa aaa gca tat agt catcag ctt aat tac gcg gaa 1440 Asp Glu Pro Leu Glu Lys Ala Tyr Ser His GlnLeu Asn Tyr Ala Glu 465 470 475 480 tgt ttc tta atg cag gac cgt cgt ggaaca att cca ttt ttt act tgg 1488 Cys Phe Leu Met Gln Asp Arg Arg Gly ThrIle Pro Phe Phe Thr Trp 485 490 495 aca cat aga agt gta gac ttt ttt aataca att gat gct gaa aag att 1536 Thr His Arg Ser Val Asp Phe Phe Asn ThrIle Asp Ala Glu Lys Ile 500 505 510 act caa ctt cca gta gtg aaa gca tatgcc ttg tct tca ggt gct tcc 1584 Thr Gln Leu Pro Val Val Lys Ala Tyr AlaLeu Ser Ser Gly Ala Ser 515 520 525 att att gaa ggt cca gga ttc aca ggagga aat tta cta ttc cta aaa 1632 Ile Ile Glu Gly Pro Gly Phe Thr Gly GlyAsn Leu Leu Phe Leu Lys 530 535 540 gaa tct agt aat tca att gct aaa tttaaa gtt aca tta aat tca gca 1680 Glu Ser Ser Asn Ser Ile Ala Lys Phe LysVal Thr Leu Asn Ser Ala 545 550 555 560 gcc ttg tta caa cga tat cgt gtaaga ata cgc tat gct tct acc act 1728 Ala Leu Leu Gln Arg Tyr Arg Val ArgIle Arg Tyr Ala Ser Thr Thr 565 570 575 aac tta cga ctt ttt gtg caa aattca aac aat gat ttt ctt gtc atc 1776 Asn Leu Arg Leu Phe Val Gln Asn SerAsn Asn Asp Phe Leu Val Ile 580 585 590 tac att aat aaa act atg aat aaagat gat gat tta aca tat caa aca 1824 Tyr Ile Asn Lys Thr Met Asn Lys AspAsp Asp Leu Thr Tyr Gln Thr 595 600 605 ttt gat ctc gca act act aat tctaat atg ggg ttc tcg ggt gat aag 1872 Phe Asp Leu Ala Thr Thr Asn Ser AsnMet Gly Phe Ser Gly Asp Lys 610 615 620 aat gaa ctt ata ata gga gca gaatct ttc gtt tct aat gaa aaa atc 1920 Asn Glu Leu Ile Ile Gly Ala Glu SerPhe Val Ser Asn Glu Lys Ile 625 630 635 640 tat ata gat aag ata gaa tttatc cca gta caa ttg taa 1959 Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val GlnLeu 645 650 2 652 PRT Bacillus thuringiensis 2 Met Asn Pro Asn Asn ArgSer Glu His Asp Thr Ile Lys Val Thr Pro 1 5 10 15 Asn Ser Glu Leu GlnThr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30 Pro Asn Ser Thr LeuGlu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45 Thr Glu Asp Ser SerThr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60 Ala Val Gly Thr GlyIle Ser Val Val Gly Gln Ile Leu Gly Val Val 65 70 75 80 Gly Val Pro PheAla Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 90 95 Asn Thr Ile TrpPro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala 100 105 110 Gln Val GluVal Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125 Lys AlaLeu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140 ValAsn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser 145 150 155160 Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser 165170 175 His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val180 185 190 Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu LeuLeu 195 200 205 Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr SerSer Glu 210 215 220 Asp Val Ala Glu Phe Tyr His Arg Gln Leu Lys Leu ThrGln Gln Tyr 225 230 235 240 Thr Asp His Cys Val Asn Trp Tyr Asn Val GlyLeu Asn Gly Leu Arg 245 250 255 Gly Ser Thr Tyr Asp Ala Trp Val Lys PheAsn Arg Phe Arg Arg Glu 260 265 270 Met Thr Leu Thr Val Leu Asp Leu IleVal Leu Phe Pro Phe Tyr Asp 275 280 285 Ile Arg Leu Tyr Ser Lys Gly ValLys Thr Glu Leu Thr Arg Asp Ile 290 295 300 Phe Thr Asp Pro Ile Phe SerLeu Asn Thr Leu Gln Glu Tyr Gly Pro 305 310 315 320 Thr Phe Leu Ser IleGlu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325 330 335 Tyr Leu Gln GlyIle Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Phe 340 345 350 Gly Lys AspSer Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365 Pro SerIle Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380 LysSer Thr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys Val 385 390 395400 Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405410 415 Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln420 425 430 Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn AsnGly 435 440 445 His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro GluThr Thr 450 455 460 Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu AsnTyr Ala Glu 465 470 475 480 Cys Phe Leu Met Gln Asp Arg Arg Gly Thr IlePro Phe Phe Thr Trp 485 490 495 Thr His Arg Ser Val Asp Phe Phe Asn ThrIle Asp Ala Glu Lys Ile 500 505 510 Thr Gln Leu Pro Val Val Lys Ala TyrAla Leu Ser Ser Gly Ala Ser 515 520 525 Ile Ile Glu Gly Pro Gly Phe ThrGly Gly Asn Leu Leu Phe Leu Lys 530 535 540 Glu Ser Ser Asn Ser Ile AlaLys Phe Lys Val Thr Leu Asn Ser Ala 545 550 555 560 Ala Leu Leu Gln ArgTyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570 575 Asn Leu Arg LeuPhe Val Gln Asn Ser Asn Asn Asp Phe Leu Val Ile 580 585 590 Tyr Ile AsnLys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605 Phe AspLeu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620 AsnGlu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile 625 630 635640 Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645 650 3 1959 DNABacillus thuringiensis CDS (1)..(1956) naturally occurring nucleotidesequence encoding a Cry3Bb2 amino acid sequence 3 atg aat cca aac aatcga agt gaa cat gat acg ata aag gtt aca cct 48 Met Asn Pro Asn Asn ArgSer Glu His Asp Thr Ile Lys Val Thr Pro 1 5 10 15 aac agt gaa ttg ccaact aac cat aat caa tat cct tta gct gac aat 96 Asn Ser Glu Leu Pro ThrAsn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30 cca aat tcg aca cta gaagaa tta aat tat aaa gaa ttt tta aga atg 144 Pro Asn Ser Thr Leu Glu GluLeu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45 act gaa gac agt tct acg gaagtg cta gac aac tct aca gta aaa gat 192 Thr Glu Asp Ser Ser Thr Glu ValLeu Asp Asn Ser Thr Val Lys Asp 50 55 60 gca gtt ggg aca gga att tct gttgta ggg cag att tta ggt gtt gta 240 Ala Val Gly Thr Gly Ile Ser Val ValGly Gln Ile Leu Gly Val Val 65 70 75 80 gga gtt cca ttt gct ggg gca ctcact tca ttt tat caa tca ttt ctt 288 Gly Val Pro Phe Ala Gly Ala Leu ThrSer Phe Tyr Gln Ser Phe Leu 85 90 95 gac act ata tgg cca agt gat gct gaccca tgg aag gct ttt atg gca 336 Asp Thr Ile Trp Pro Ser Asp Ala Asp ProTrp Lys Ala Phe Met Ala 100 105 110 caa gtt gaa gta ctg ata gat aag aaaata gag gag tat gct aaa agt 384 Gln Val Glu Val Leu Ile Asp Lys Lys IleGlu Glu Tyr Ala Lys Ser 115 120 125 aaa gct ctt gca gag tta cag ggt cttcaa aat aat ttc gaa gat tat 432 Lys Ala Leu Ala Glu Leu Gln Gly Leu GlnAsn Asn Phe Glu Asp Tyr 130 135 140 gtt aat gcg tta aat tcc tgg aag aaaaca cct tta agt ttg cga agt 480 Val Asn Ala Leu Asn Ser Trp Lys Lys ThrPro Leu Ser Leu Arg Ser 145 150 155 160 aaa aga agc caa gat cga ata agggaa ctt ttt tct caa gca gaa agt 528 Lys Arg Ser Gln Asp Arg Ile Arg GluLeu Phe Ser Gln Ala Glu Ser 165 170 175 cat ttt cgt aat tcc atg ccg tcattt gca gtt tcc aaa ttc gaa gtg 576 His Phe Arg Asn Ser Met Pro Ser PheAla Val Ser Lys Phe Glu Val 180 185 190 ctg ttt cta cca aca tat gca caagct gca aat aca cat tta ttg cta 624 Leu Phe Leu Pro Thr Tyr Ala Gln AlaAla Asn Thr His Leu Leu Leu 195 200 205 tta aaa gat gct caa gtt ttt ggagaa gaa tgg gga tat tct tca gaa 672 Leu Lys Asp Ala Gln Val Phe Gly GluGlu Trp Gly Tyr Ser Ser Glu 210 215 220 gat gtt gct gaa ttt tat cat agacaa tta aaa ctt acg caa caa tac 720 Asp Val Ala Glu Phe Tyr His Arg GlnLeu Lys Leu Thr Gln Gln Tyr 225 230 235 240 act gac cat tgt gtc aat tggtat aat gtt gga tta aat ggt tta aga 768 Thr Asp His Cys Val Asn Trp TyrAsn Val Gly Leu Asn Gly Leu Arg 245 250 255 ggt tca act tat gat gca tgggtc aaa ttt aac cgt ttt cgc aga gaa 816 Gly Ser Thr Tyr Asp Ala Trp ValLys Phe Asn Arg Phe Arg Arg Glu 260 265 270 atg act tta act gta tta gatcta att gta ctt ttc cca ttt tat gat 864 Met Thr Leu Thr Val Leu Asp LeuIle Val Leu Phe Pro Phe Tyr Asp 275 280 285 gtt cgg tta tac tca aaa ggtgtt aaa aca gaa cta aca aga gac att 912 Val Arg Leu Tyr Ser Lys Gly ValLys Thr Glu Leu Thr Arg Asp Ile 290 295 300 ttt acg gat cca att ttt tcactc aat act ctt cag gag tat gga cca 960 Phe Thr Asp Pro Ile Phe Ser LeuAsn Thr Leu Gln Glu Tyr Gly Pro 305 310 315 320 act ttt ttg agt ata gaaaac tct att cga aaa cct cat tta ttt gat 1008 Thr Phe Leu Ser Ile Glu AsnSer Ile Arg Lys Pro His Leu Phe Asp 325 330 335 tat tta cag ggt att gaattt cat acg cgt ctt caa cct ggt tac tct 1056 Tyr Leu Gln Gly Ile Glu PheHis Thr Arg Leu Gln Pro Gly Tyr Ser 340 345 350 ggg aaa gat tct ttc aattat tgg tct ggt aat tat gta gaa act aga 1104 Gly Lys Asp Ser Phe Asn TyrTrp Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365 cct agt ata gga tct agtaag aca att act tcc cca ttt tat gga gat 1152 Pro Ser Ile Gly Ser Ser LysThr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380 aaa tct act gaa cct gtacaa aag tta agc ttt gat gga caa aaa gtt 1200 Lys Ser Thr Glu Pro Val GlnLys Leu Ser Phe Asp Gly Gln Lys Val 385 390 395 400 tat cga act ata gctaat aca gac gta gcg gct tgg ccg aat ggc aag 1248 Tyr Arg Thr Ile Ala AsnThr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410 415 ata tat ttt ggt gttacg aaa gtt gat ttt agt caa tat gat gat caa 1296 Ile Tyr Phe Gly Val ThrLys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425 430 aaa aat gaa act agtaca caa aca tat gat tca aaa aga aac aat ggc 1344 Lys Asn Glu Thr Ser ThrGln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445 cat gta ggt gca caggat tct att gac caa tta cca cca gaa aca aca 1392 His Val Gly Ala Gln AspSer Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455 460 gat gaa cca ctt gaaaaa gca tat agt cat cag ctt aat tac gcg gaa 1440 Asp Glu Pro Leu Glu LysAla Tyr Ser His Gln Leu Asn Tyr Ala Glu 465 470 475 480 tgt ttc tta atgcag gac cgt cgt gga aca att cca ttt ttt act tgg 1488 Cys Phe Leu Met GlnAsp Arg Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495 aca cat aga agtgta gac ttt ttt aat aca att gat gct gaa aag att 1536 Thr His Arg Ser ValAsp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510 act caa ctt ccagta gtg aaa gca tat gcc ttg tct tca ggt gct tcc 1584 Thr Gln Leu Pro ValVal Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525 att att gaa ggtcca gga ttc aca gga gga aat tta cta ttc cta aaa 1632 Ile Ile Glu Gly ProGly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535 540 gaa tct agt aattca att gct aaa ttt aaa gtt aca tta aat tca gca 1680 Glu Ser Ser Asn SerIle Ala Lys Phe Lys Val Thr Leu Asn Ser Ala 545 550 555 560 gcc ttg ttacaa cga tat cgt gta aga ata cgc tat gct tct acc act 1728 Ala Leu Leu GlnArg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570 575 aac tta cgactt ttt gtg caa aat tca aac aat gat ttt att gtc atc 1776 Asn Leu Arg LeuPhe Val Gln Asn Ser Asn Asn Asp Phe Ile Val Ile 580 585 590 tac att aataaa act atg aat ata gat gat gat tta aca tat caa aca 1824 Tyr Ile Asn LysThr Met Asn Ile Asp Asp Asp Leu Thr Tyr Gln Thr 595 600 605 ttt gat ctcgca act act aat tct aat atg ggg ttc tcg ggt gat acg 1872 Phe Asp Leu AlaThr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp Thr 610 615 620 aat gaa cttata ata gga gca gaa tct ttc gtt tct aat gaa aaa atc 1920 Asn Glu Leu IleIle Gly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile 625 630 635 640 tat atagat aag ata gaa ttt atc cca gta caa ttg taa 1959 Tyr Ile Asp Lys Ile GluPhe Ile Pro Val Gln Leu 645 650 4 652 PRT Bacillus thuringiensis 4 MetAsn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr Pro 1 5 10 15Asn Ser Glu Leu Pro Thr Asn His Asn Gln Tyr Pro Leu Ala Asp Asn 20 25 30Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg Met 35 40 45Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys Asp 50 55 60Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val Val 65 70 7580 Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe Leu 85 9095 Asp Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met Ala 100105 110 Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys Ser115 120 125 Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu AspTyr 130 135 140 Val Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser LeuArg Ser 145 150 155 160 Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe SerGln Ala Glu Ser 165 170 175 His Phe Arg Asn Ser Met Pro Ser Phe Ala ValSer Lys Phe Glu Val 180 185 190 Leu Phe Leu Pro Thr Tyr Ala Gln Ala AlaAsn Thr His Leu Leu Leu 195 200 205 Leu Lys Asp Ala Gln Val Phe Gly GluGlu Trp Gly Tyr Ser Ser Glu 210 215 220 Asp Val Ala Glu Phe Tyr His ArgGln Leu Lys Leu Thr Gln Gln Tyr 225 230 235 240 Thr Asp His Cys Val AsnTrp Tyr Asn Val Gly Leu Asn Gly Leu Arg 245 250 255 Gly Ser Thr Tyr AspAla Trp Val Lys Phe Asn Arg Phe Arg Arg Glu 260 265 270 Met Thr Leu ThrVal Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr Asp 275 280 285 Val Arg LeuTyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp Ile 290 295 300 Phe ThrAsp Pro Ile Phe Ser Leu Asn Thr Leu Gln Glu Tyr Gly Pro 305 310 315 320Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe Asp 325 330335 Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro Gly Tyr Ser 340345 350 Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr Arg355 360 365 Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr GlyAsp 370 375 380 Lys Ser Thr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly GlnLys Val 385 390 395 400 Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala Ala TrpPro Asn Gly Lys 405 410 415 Ile Tyr Phe Gly Val Thr Lys Val Asp Phe SerGln Tyr Asp Asp Gln 420 425 430 Lys Asn Glu Thr Ser Thr Gln Thr Tyr AspSer Lys Arg Asn Asn Gly 435 440 445 His Val Gly Ala Gln Asp Ser Ile AspGln Leu Pro Pro Glu Thr Thr 450 455 460 Asp Glu Pro Leu Glu Lys Ala TyrSer His Gln Leu Asn Tyr Ala Glu 465 470 475 480 Cys Phe Leu Met Gln AspArg Arg Gly Thr Ile Pro Phe Phe Thr Trp 485 490 495 Thr His Arg Ser ValAsp Phe Phe Asn Thr Ile Asp Ala Glu Lys Ile 500 505 510 Thr Gln Leu ProVal Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala Ser 515 520 525 Ile Ile GluGly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu Lys 530 535 540 Glu SerSer Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser Ala 545 550 555 560Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr Thr 565 570575 Asn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Ile Val Ile 580585 590 Tyr Ile Asn Lys Thr Met Asn Ile Asp Asp Asp Leu Thr Tyr Gln Thr595 600 605 Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly AspThr 610 615 620 Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn GluLys Ile 625 630 635 640 Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu645 650 5 1962 DNA Artificial Sequence Description of ArtificialSequence synthetic or non-naturally occurring nucleotide sequenceencoding a Cry3Bb amino acid sequence 5 atg aac cct aac aat cgt tcc gaacac gac acc atc aag gtt act cca 48 Met Asn Pro Asn Asn Arg Ser Glu HisAsp Thr Ile Lys Val Thr Pro 1 5 10 15 aac tct gag ttg caa act aat cacaac cag tac cca ttg gct gac aat 96 Asn Ser Glu Leu Gln Thr Asn His AsnGln Tyr Pro Leu Ala Asp Asn 20 25 30 cct aac agt act ctt gag gaa ctt aactac aag gag ttt ctc cgg atg 144 Pro Asn Ser Thr Leu Glu Glu Leu Asn TyrLys Glu Phe Leu Arg Met 35 40 45 acc gaa gat agc tcc act gag gtt ctc gataac tct aca gtg aag gac 192 Thr Glu Asp Ser Ser Thr Glu Val Leu Asp AsnSer Thr Val Lys Asp 50 55 60 gct gtt gga act ggc att agc gtt gtg gga cagatt ctt gga gtg gtt 240 Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln IleLeu Gly Val Val 65 70 75 80 ggt gtt cca ttc gct gga gct ttg acc agc ttctac cag tcc ttt ctc 288 Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe TyrGln Ser Phe Leu 85 90 95 aac acc atc tgg cct tca gat gct gat ccc tgg aaggct ttc atg gcc 336 Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys AlaPhe Met Ala 100 105 110 caa gtg gaa gtc ttg atc gat aag aag atc gaa gagtat gcc aag tct 384 Gln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu TyrAla Lys Ser 115 120 125 aaa gcc ttg gct gag ttg caa ggt ttg cag aac aacttc gag gat tac 432 Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn PheGlu Asp Tyr 130 135 140 gtc aac gca ctc aac agc tgg aag aaa act ccc ttgagt ctc agg tct 480 Val Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu SerLeu Arg Ser 145 150 155 160 aag cgt tcc cag gac cgt att cgt gaa ctt ttcagc caa gcc gaa tcc 528 Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe SerGln Ala Glu Ser 165 170 175 cac ttc aga aac tcc atg cct agc ttt gcc gtttct aag ttc gag gtg 576 His Phe Arg Asn Ser Met Pro Ser Phe Ala Val SerLys Phe Glu Val 180 185 190 ctc ttc ttg cca aca tac gca caa gct gcc aacact cat ctc ttg ctt 624 Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn ThrHis Leu Leu Leu 195 200 205 ctc aaa gac gct cag gtg ttt ggt gag gaa tggggt tac tcc agt gaa 672 Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp GlyTyr Ser Ser Glu 210 215 220 gat gtt gcc gag ttc tac cat agg cag ctc aagttg act caa cag tac 720 Asp Val Ala Glu Phe Tyr His Arg Gln Leu Lys LeuThr Gln Gln Tyr 225 230 235 240 aca gac cac tgc gtc aac tgg tac aac gttggg ctc aat ggt ctt aga 768 Thr Asp His Cys Val Asn Trp Tyr Asn Val GlyLeu Asn Gly Leu Arg 245 250 255 gga tct acc tac gac gca tgg gtg aag ttcaac agg ttt cgt aga gag 816 Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe AsnArg Phe Arg Arg Glu 260 265 270 atg acc ttg act gtg ctc gat ctt atc gttctc ttt cca ttc tac gac 864 Met Thr Leu Thr Val Leu Asp Leu Ile Val LeuPhe Pro Phe Tyr Asp 275 280 285 att cgt ctt tac tcc aaa ggc gtt aag acagag ctg acc aga gac atc 912 Ile Arg Leu Tyr Ser Lys Gly Val Lys Thr GluLeu Thr Arg Asp Ile 290 295 300 ttc acc gat ccc atc ttc tca ctt aac accctg cag gaa tac ggt cca 960 Phe Thr Asp Pro Ile Phe Ser Leu Asn Thr LeuGln Glu Tyr Gly Pro 305 310 315 320 act ttt ctc tcc att gag aac agc atcagg aag cct cac ctc ttc gac 1008 Thr Phe Leu Ser Ile Glu Asn Ser Ile ArgLys Pro His Leu Phe Asp 325 330 335 tat ctg caa ggc att gag ttt cac accagg ttg caa cct ggt tac ttc 1056 Tyr Leu Gln Gly Ile Glu Phe His Thr ArgLeu Gln Pro Gly Tyr Phe 340 345 350 ggt aag gat tcc ttc aac tac tgg agcgga aac tac gtt gaa acc aga 1104 Gly Lys Asp Ser Phe Asn Tyr Trp Ser GlyAsn Tyr Val Glu Thr Arg 355 360 365 cca tcc atc gga tct agc aag acc atcact tct cca ttc tac ggt gac 1152 Pro Ser Ile Gly Ser Ser Lys Thr Ile ThrSer Pro Phe Tyr Gly Asp 370 375 380 aag agc act gag cca gtg cag aag ttgagc ttc gat ggg cag aag gtg 1200 Lys Ser Thr Glu Pro Val Gln Lys Leu SerPhe Asp Gly Gln Lys Val 385 390 395 400 tat aga acc atc gcc aat acc gatgtt gca gct tgg cct aat ggc aag 1248 Tyr Arg Thr Ile Ala Asn Thr Asp ValAla Ala Trp Pro Asn Gly Lys 405 410 415 gtc tac ctt gga gtt act aaa gtggac ttc tcc caa tac gac gat cag 1296 Val Tyr Leu Gly Val Thr Lys Val AspPhe Ser Gln Tyr Asp Asp Gln 420 425 430 aag aac gag aca tct act caa acctac gat agt aag agg aac aat ggc 1344 Lys Asn Glu Thr Ser Thr Gln Thr TyrAsp Ser Lys Arg Asn Asn Gly 435 440 445 cat gtt tcc gca caa gac tcc attgac caa ctt cca cct gaa acc act 1392 His Val Ser Ala Gln Asp Ser Ile AspGln Leu Pro Pro Glu Thr Thr 450 455 460 gat gaa cca ttg gag aag gct tacagt cac caa ctt aac tac gcc gaa 1440 Asp Glu Pro Leu Glu Lys Ala Tyr SerHis Gln Leu Asn Tyr Ala Glu 465 470 475 480 tgc ttt ctc atg caa gac aggcgt ggc acc att ccg ttc ttt aca tgg 1488 Cys Phe Leu Met Gln Asp Arg ArgGly Thr Ile Pro Phe Phe Thr Trp 485 490 495 act cac agg tct gtc gac ttcttt aac act atc gac gct gag aag att 1536 Thr His Arg Ser Val Asp Phe PheAsn Thr Ile Asp Ala Glu Lys Ile 500 505 510 acc caa ctt ccc gtg gtc aaggct tat gcc ttg tcc agc gga gct tcc 1584 Thr Gln Leu Pro Val Val Lys AlaTyr Ala Leu Ser Ser Gly Ala Ser 515 520 525 atc att gaa ggt cca ggc ttcacc ggt ggc aac ttg ctc ttc ctt aag 1632 Ile Ile Glu Gly Pro Gly Phe ThrGly Gly Asn Leu Leu Phe Leu Lys 530 535 540 gag tcc agc aac tcc atc gccaag ttc aaa gtg aca ctt aac tca gca 1680 Glu Ser Ser Asn Ser Ile Ala LysPhe Lys Val Thr Leu Asn Ser Ala 545 550 555 560 gcc ttg ctc caa cgt tacagg gtt cgt atc aga tac gca agc act acc 1728 Ala Leu Leu Gln Arg Tyr ArgVal Arg Ile Arg Tyr Ala Ser Thr Thr 565 570 575 aat ctt cgc ctc ttt gtccag aac agc aac aat gat ttc ctt gtc atc 1776 Asn Leu Arg Leu Phe Val GlnAsn Ser Asn Asn Asp Phe Leu Val Ile 580 585 590 tac atc aac aag act atgaac aaa gac gat gac ctc acc tac aac aca 1824 Tyr Ile Asn Lys Thr Met AsnLys Asp Asp Asp Leu Thr Tyr Asn Thr 595 600 605 ttc gat ctt gcc act accaat agt aac atg gga ttc tct ggt gac aag 1872 Phe Asp Leu Ala Thr Thr AsnSer Asn Met Gly Phe Ser Gly Asp Lys 610 615 620 aac gag ctg atc ata ggtgct gag agc ttt gtc tct aat gag aag att 1920 Asn Glu Leu Ile Ile Gly AlaGlu Ser Phe Val Ser Asn Glu Lys Ile 625 630 635 640 tac ata gac aag atcgag ttc att cca gtt caa ctc taatag 1962 Tyr Ile Asp Lys Ile Glu Phe IlePro Val Gln Leu 645 650 6 652 PRT Artificial Sequence Description ofArtificial Sequence synthetic or non-naturally occurring amino acidsequence encoded by SEQ ID NO5 6 Met Asn Pro Asn Asn Arg Ser Glu His AspThr Ile Lys Val Thr Pro 1 5 10 15 Asn Ser Glu Leu Gln Thr Asn His AsnGln Tyr Pro Leu Ala Asp Asn 20 25 30 Pro Asn Ser Thr Leu Glu Glu Leu AsnTyr Lys Glu Phe Leu Arg Met 35 40 45 Thr Glu Asp Ser Ser Thr Glu Val LeuAsp Asn Ser Thr Val Lys Asp 50 55 60 Ala Val Gly Thr Gly Ile Ser Val ValGly Gln Ile Leu Gly Val Val 65 70 75 80 Gly Val Pro Phe Ala Gly Ala LeuThr Ser Phe Tyr Gln Ser Phe Leu 85 90 95 Asn Thr Ile Trp Pro Ser Asp AlaAsp Pro Trp Lys Ala Phe Met Ala 100 105 110 Gln Val Glu Val Leu Ile AspLys Lys Ile Glu Glu Tyr Ala Lys Ser 115 120 125 Lys Ala Leu Ala Glu LeuGln Gly Leu Gln Asn Asn Phe Glu Asp Tyr 130 135 140 Val Asn Ala Leu AsnSer Trp Lys Lys Thr Pro Leu Ser Leu Arg Ser 145 150 155 160 Lys Arg SerGln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu Ser 165 170 175 His PheArg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu Val 180 185 190 LeuPhe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu Leu 195 200 205Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser Glu 210 215220 Asp Val Ala Glu Phe Tyr His Arg Gln Leu Lys Leu Thr Gln Gln Tyr 225230 235 240 Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu Asn Gly LeuArg 245 250 255 Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe ArgArg Glu 260 265 270 Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe ProPhe Tyr Asp 275 280 285 Ile Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu LeuThr Arg Asp Ile 290 295 300 Phe Thr Asp Pro Ile Phe Ser Leu Asn Thr LeuGln Glu Tyr Gly Pro 305 310 315 320 Thr Phe Leu Ser Ile Glu Asn Ser IleArg Lys Pro His Leu Phe Asp 325 330 335 Tyr Leu Gln Gly Ile Glu Phe HisThr Arg Leu Gln Pro Gly Tyr Phe 340 345 350 Gly Lys Asp Ser Phe Asn TyrTrp Ser Gly Asn Tyr Val Glu Thr Arg 355 360 365 Pro Ser Ile Gly Ser SerLys Thr Ile Thr Ser Pro Phe Tyr Gly Asp 370 375 380 Lys Ser Thr Glu ProVal Gln Lys Leu Ser Phe Asp Gly Gln Lys Val 385 390 395 400 Tyr Arg ThrIle Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly Lys 405 410 415 Val TyrLeu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp Gln 420 425 430 LysAsn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn Gly 435 440 445His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr Thr 450 455460 Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala Glu 465470 475 480 Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe ThrTrp 485 490 495 Thr His Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala GluLys Ile 500 505 510 Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser SerGly Ala Ser 515 520 525 Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn LeuLeu Phe Leu Lys 530 535 540 Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys ValThr Leu Asn Ser Ala 545 550 555 560 Ala Leu Leu Gln Arg Tyr Arg Val ArgIle Arg Tyr Ala Ser Thr Thr 565 570 575 Asn Leu Arg Leu Phe Val Gln AsnSer Asn Asn Asp Phe Leu Val Ile 580 585 590 Tyr Ile Asn Lys Thr Met AsnLys Asp Asp Asp Leu Thr Tyr Asn Thr 595 600 605 Phe Asp Leu Ala Thr ThrAsn Ser Asn Met Gly Phe Ser Gly Asp Lys 610 615 620 Asn Glu Leu Ile IleGly Ala Glu Ser Phe Val Ser Asn Glu Lys Ile 625 630 635 640 Tyr Ile AspLys Ile Glu Phe Ile Pro Val Gln Leu 645 650 7 1989 DNA ArtificialSequence Description of Artificial Sequence non- naturally occurringnucleotide sequence encoding a variant Cry3Bb amino acid sequence v112317 cc atg gca aac cct aac aat cgt tcc gaa cac gac acc atc aag gtt 47 MetAla Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val 1 5 10 15 actcca aac tct gag ttg caa act aat cac aac cag tac cca ttg gct 95 Thr ProAsn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala 20 25 30 gac aatcct aac agt act ctt gag gaa ctt aac tac aag gag ttt ctc 143 Asp Asn ProAsn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu 35 40 45 cgg atg accgaa gat agc tcc act gag gtt ctc gat aac tct aca gtg 191 Arg Met Thr GluAsp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val 50 55 60 aag gac gct gttgga act ggc att agc gtt gtg gga cag att ctt gga 239 Lys Asp Ala Val GlyThr Gly Ile Ser Val Val Gly Gln Ile Leu Gly 65 70 75 gtg gtt ggt gtt ccattc gct gga gct ttg acc agc ttc tac cag tcc 287 Val Val Gly Val Pro PheAla Gly Ala Leu Thr Ser Phe Tyr Gln Ser 80 85 90 95 ttt ctc aac acc atctgg cct tca gat gct gat ccc tgg aag gct ttc 335 Phe Leu Asn Thr Ile TrpPro Ser Asp Ala Asp Pro Trp Lys Ala Phe 100 105 110 atg gcc caa gtg gaagtc ttg atc gat aag aag atc gaa gag tat gcc 383 Met Ala Gln Val Glu ValLeu Ile Asp Lys Lys Ile Glu Glu Tyr Ala 115 120 125 aag tct aaa gcc ttggct gag ttg caa ggt ttg cag aac aac ttc gag 431 Lys Ser Lys Ala Leu AlaGlu Leu Gln Gly Leu Gln Asn Asn Phe Glu 130 135 140 gat tac gtc aac gcactc aac agc tgg aag aaa act ccc ttg agt ctc 479 Asp Tyr Val Asn Ala LeuAsn Ser Trp Lys Lys Thr Pro Leu Ser Leu 145 150 155 agg tct aag cgt tcccag gac cgt att cgt gaa ctt ttc agc caa gcc 527 Arg Ser Lys Arg Ser GlnAsp Arg Ile Arg Glu Leu Phe Ser Gln Ala 160 165 170 175 gaa tcc cac ttcaga aac tcc atg cct agc ttt gcc gtt tct aag ttc 575 Glu Ser His Phe ArgAsn Ser Met Pro Ser Phe Ala Val Ser Lys Phe 180 185 190 gag gtg ctc ttcttg cca aca tac gca caa gct gcc aac act cat ctc 623 Glu Val Leu Phe LeuPro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu 195 200 205 ttg ctt ctc aaagac gct cag gtg ttt ggt gag gaa tgg ggt tac tcc 671 Leu Leu Leu Lys AspAla Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser 210 215 220 agt gaa gat gttgcc gag ttc tac cgt agg cag ctc aag ttg act caa 719 Ser Glu Asp Val AlaGlu Phe Tyr Arg Arg Gln Leu Lys Leu Thr Gln 225 230 235 cag tac aca gaccac tgc gtc aac tgg tac aac gtt ggg ctc aat ggt 767 Gln Tyr Thr Asp HisCys Val Asn Trp Tyr Asn Val Gly Leu Asn Gly 240 245 250 255 ctt aga ggatct acc tac gac gca tgg gtg aag ttc aac agg ttt cgt 815 Leu Arg Gly SerThr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg 260 265 270 aga gag atgacc ttg act gtg ctc gat ctt atc gtt ctc ttt cca ttc 863 Arg Glu Met ThrLeu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe 275 280 285 tac gac attcgt ctt tac tcc aaa ggc gtt aag aca gag ctg acc aga 911 Tyr Asp Ile ArgLeu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg 290 295 300 gac atc ttcacc gat ccc atc ttc cta ctt acg acc ctg cag aaa tac 959 Asp Ile Phe ThrAsp Pro Ile Phe Leu Leu Thr Thr Leu Gln Lys Tyr 305 310 315 ggt cca actttt ctc tcc att gag aac agc atc agg aag cct cac ctc 1007 Gly Pro Thr PheLeu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu 320 325 330 335 ttc gactat ctg caa ggc att gag ttt cac acc agg ttg caa cct ggt 1055 Phe Asp TyrLeu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro Gly 340 345 350 tac ttcggt aag gat tcc ttc aac tac tgg agc gga aac tac gtt gaa 1103 Tyr Phe GlyLys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu 355 360 365 acc agacca tcc atc gga tct agc aag acc atc act tct cca ttc tac 1151 Thr Arg ProSer Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr 370 375 380 ggt gacaag agc act gag cca gtg cag aag ttg agc ttc gat ggg cag 1199 Gly Asp LysSer Thr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly Gln 385 390 395 aag gtgtat aga acc atc gcc aat acc gat gtt gca gct tgg cct aat 1247 Lys Val TyrArg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn 400 405 410 415 ggcaag gtc tac ctt gga gtt act aaa gtg gac ttc tcc caa tac gac 1295 Gly LysVal Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp 420 425 430 gatcag aag aac gag aca tct act caa acc tac gat agt aag agg aac 1343 Asp GlnLys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn 435 440 445 aatggc cat gtt tcc gca caa gac tcc att gac caa ctt cca cct gaa 1391 Asn GlyHis Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu 450 455 460 accact gat gaa cca ttg gag aag gct tac agt cac caa ctt aac tac 1439 Thr ThrAsp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr 465 470 475 gccgaa tgc ttt ctc atg caa gac agg cgt ggc acc att ccg ttc ttt 1487 Ala GluCys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe 480 485 490 495aca tgg act cac agg tct gtc gac ttc ttt aac act atc gac gct gag 1535 ThrTrp Thr His Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala Glu 500 505 510aag att acc caa ctt ccc gtg gtc aag gct tat gcc ttg tcc agc gga 1583 LysIle Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly 515 520 525gct tcc atc att gaa ggt cca ggc ttc acc ggt ggc aac ttg ctc ttc 1631 AlaSer Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe 530 535 540ctt aag gag tcc agc aac tcc atc gcc aag ttc aaa gtg aca ctt aac 1679 LeuLys Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn 545 550 555tca gca gcc ttg ctc caa cgt tac agg gtt cgt atc aga tac gca agc 1727 SerAla Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser 560 565 570575 act acc aat ctt cgc ctc ttt gtc cag aac agc aac aat gat ttc ctt 1775Thr Thr Asn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu 580 585590 gtc atc tac atc aac aag act atg aac aaa gac gat gac ctc acc tac 1823Val Ile Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr 595 600605 caa aca ttc gat ctt gcc act acc aat agt aac atg gga ttc tct ggt 1871Gln Thr Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly 610 615620 gac aag aac gag ctg atc ata ggt gct gag agc ttt gtc tct aat gag 1919Asp Lys Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu 625 630635 aag att tac ata gac aag atc gag ttc att cca gtt caa ctc 1961 Lys IleTyr Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 640 645 650 taatagatcccccgggctgc aggaattc 1989 8 653 PRT Artificial Sequence Description ofArtificial Sequence non- naturally occurring amino acid sequence encodedby SEQ ID NO7 8 Met Ala Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile LysVal Thr 1 5 10 15 Pro Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr ProLeu Ala Asp 20 25 30 Asn Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys GluPhe Leu Arg 35 40 45 Met Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn SerThr Val Lys 50 55 60 Asp Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln IleLeu Gly Val 65 70 75 80 Val Gly Val Pro Phe Ala Gly Ala Leu Thr Ser PheTyr Gln Ser Phe 85 90 95 Leu Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro TrpLys Ala Phe Met 100 105 110 Ala Gln Val Glu Val Leu Ile Asp Lys Lys IleGlu Glu Tyr Ala Lys 115 120 125 Ser Lys Ala Leu Ala Glu Leu Gln Gly LeuGln Asn Asn Phe Glu Asp 130 135 140 Tyr Val Asn Ala Leu Asn Ser Trp LysLys Thr Pro Leu Ser Leu Arg 145 150 155 160 Ser Lys Arg Ser Gln Asp ArgIle Arg Glu Leu Phe Ser Gln Ala Glu 165 170 175 Ser His Phe Arg Asn SerMet Pro Ser Phe Ala Val Ser Lys Phe Glu 180 185 190 Val Leu Phe Leu ProThr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu 195 200 205 Leu Leu Lys AspAla Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser 210 215 220 Glu Asp ValAla Glu Phe Tyr Arg Arg Gln Leu Lys Leu Thr Gln Gln 225 230 235 240 TyrThr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu Asn Gly Leu 245 250 255Arg Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg 260 265270 Glu Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr 275280 285 Asp Ile Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp290 295 300 Ile Phe Thr Asp Pro Ile Phe Leu Leu Thr Thr Leu Gln Lys TyrGly 305 310 315 320 Pro Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys ProHis Leu Phe 325 330 335 Asp Tyr Leu Gln Gly Ile Glu Phe His Thr Arg LeuGln Pro Gly Tyr 340 345 350 Phe Gly Lys Asp Ser Phe Asn Tyr Trp Ser GlyAsn Tyr Val Glu Thr 355 360 365 Arg Pro Ser Ile Gly Ser Ser Lys Thr IleThr Ser Pro Phe Tyr Gly 370 375 380 Asp Lys Ser Thr Glu Pro Val Gln LysLeu Ser Phe Asp Gly Gln Lys 385 390 395 400 Val Tyr Arg Thr Ile Ala AsnThr Asp Val Ala Ala Trp Pro Asn Gly 405 410 415 Lys Val Tyr Leu Gly ValThr Lys Val Asp Phe Ser Gln Tyr Asp Asp 420 425 430 Gln Lys Asn Glu ThrSer Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn 435 440 445 Gly His Val SerAla Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr 450 455 460 Thr Asp GluPro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala 465 470 475 480 GluCys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr 485 490 495Trp Thr His Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys 500 505510 Ile Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala 515520 525 Ser Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu530 535 540 Lys Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu AsnSer 545 550 555 560 Ala Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg TyrAla Ser Thr 565 570 575 Thr Asn Leu Arg Leu Phe Val Gln Asn Ser Asn AsnAsp Phe Leu Val 580 585 590 Ile Tyr Ile Asn Lys Thr Met Asn Lys Asp AspAsp Leu Thr Tyr Gln 595 600 605 Thr Phe Asp Leu Ala Thr Thr Asn Ser AsnMet Gly Phe Ser Gly Asp 610 615 620 Lys Asn Glu Leu Ile Ile Gly Ala GluSer Phe Val Ser Asn Glu Lys 625 630 635 640 Ile Tyr Ile Asp Lys Ile GluPhe Ile Pro Val Gln Leu 645 650 9 1984 DNA Artificial SequenceDescription of Artificial Sequence non-naturally occurring nucleotidesequence encoding a Cry3Bb variant 11231mv1 amino acid sequence 9 cc atggcc aac ccc aac aat cgc tcc gag cac gac acg atc aag gtc 47 Met Ala AsnPro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val 1 5 10 15 acc ccc aactcc gag ctc cag acc aac cac aac cag tac ccg ctg gcc 95 Thr Pro Asn SerGlu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala 20 25 30 gac aac ccc aactcc acc ctg gaa gag ctg aac tac aag gag ttc ctg 143 Asp Asn Pro Asn SerThr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu 35 40 45 cgc atg acc gag gactcc tcc acg gag gtc ctg gac aac tcc acc gtc 191 Arg Met Thr Glu Asp SerSer Thr Glu Val Leu Asp Asn Ser Thr Val 50 55 60 aag gac gcc gtc ggg accggc atc tcc gtc gtt ggg cag atc ctg ggc 239 Lys Asp Ala Val Gly Thr GlyIle Ser Val Val Gly Gln Ile Leu Gly 65 70 75 gtc gtt ggc gtc ccc ttc gcaggt gct ctc acc tcc ttc tac cag tcc 287 Val Val Gly Val Pro Phe Ala GlyAla Leu Thr Ser Phe Tyr Gln Ser 80 85 90 95 ttc ctg aac acc atc tgg ccctcc gac gcc gac ccc tgg aag gcc ttc 335 Phe Leu Asn Thr Ile Trp Pro SerAsp Ala Asp Pro Trp Lys Ala Phe 100 105 110 atg gcc caa gtc gaa gtc ctgatc gac aag aag atc gag gag tac gcc 383 Met Ala Gln Val Glu Val Leu IleAsp Lys Lys Ile Glu Glu Tyr Ala 115 120 125 aag tcc aag gcc ctg gcc gagctg caa ggc ctg caa aac aac ttc gag 431 Lys Ser Lys Ala Leu Ala Glu LeuGln Gly Leu Gln Asn Asn Phe Glu 130 135 140 gac tac gtc aac gcg ctg aactcc tgg aag aag acg cct ctg tcc ctg 479 Asp Tyr Val Asn Ala Leu Asn SerTrp Lys Lys Thr Pro Leu Ser Leu 145 150 155 cgc tcc aag cgc tcc cag ggccgc atc cgc gag ctg ttc tcc cag gcc 527 Arg Ser Lys Arg Ser Gln Gly ArgIle Arg Glu Leu Phe Ser Gln Ala 160 165 170 175 gag tcc cac ttc cgc aactcc atg ccg tcc ttc gcc gtc tcc aag ttc 575 Glu Ser His Phe Arg Asn SerMet Pro Ser Phe Ala Val Ser Lys Phe 180 185 190 gag gtc ctg ttc ctg cccacc tac gcc cag gct gcc aac acc cac ctc 623 Glu Val Leu Phe Leu Pro ThrTyr Ala Gln Ala Ala Asn Thr His Leu 195 200 205 ctg ttg ctg aag gac gcccag gtc ttc ggc gag gaa tgg ggc tac tcc 671 Leu Leu Leu Lys Asp Ala GlnVal Phe Gly Glu Glu Trp Gly Tyr Ser 210 215 220 tcg gag gac gtc gcc gagttc tac cgt cgc cag ctg aag ctg acc caa 719 Ser Glu Asp Val Ala Glu PheTyr Arg Arg Gln Leu Lys Leu Thr Gln 225 230 235 cag tac acc gac cac tgcgtc aac tgg tac aac gtc ggc ctg aac ggc 767 Gln Tyr Thr Asp His Cys ValAsn Trp Tyr Asn Val Gly Leu Asn Gly 240 245 250 255 ctg agg ggc tcc acctac gac gca tgg gtc aag ttc aac cgc ttc cgc 815 Leu Arg Gly Ser Thr TyrAsp Ala Trp Val Lys Phe Asn Arg Phe Arg 260 265 270 agg gag atg acc ctgacc gtc ctg gac ctg atc gtc ctg ttc ccc ttc 863 Arg Glu Met Thr Leu ThrVal Leu Asp Leu Ile Val Leu Phe Pro Phe 275 280 285 tac gac atc cgc ctgtac tcc aag ggc gtc aag acc gag ctg acc cgc 911 Tyr Asp Ile Arg Leu TyrSer Lys Gly Val Lys Thr Glu Leu Thr Arg 290 295 300 gac atc ttc acg gacccc atc ttc ctg ctc acg acc ctc cag aag tac 959 Asp Ile Phe Thr Asp ProIle Phe Leu Leu Thr Thr Leu Gln Lys Tyr 305 310 315 ggt ccc acc ttc ctgtcc atc gag aac tcc atc cgc aag ccc cac ctg 1007 Gly Pro Thr Phe Leu SerIle Glu Asn Ser Ile Arg Lys Pro His Leu 320 325 330 335 ttc gac tac ctccag ggc atc gag ttc cac acg cgc ctg agg cca ggc 1055 Phe Asp Tyr Leu GlnGly Ile Glu Phe His Thr Arg Leu Arg Pro Gly 340 345 350 tac ttc ggc aaggac tcc ttc aac tac tgg tcc ggc aac tac gtc gag 1103 Tyr Phe Gly Lys AspSer Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu 355 360 365 acc agg ccc tccatc ggc tcc tcg aag acg atc acc tcc cct ttc tac 1151 Thr Arg Pro Ser IleGly Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr 370 375 380 ggc gac aag tccacc gag ccc gtc cag aag ctg tcc ttc gac ggc cag 1199 Gly Asp Lys Ser ThrGlu Pro Val Gln Lys Leu Ser Phe Asp Gly Gln 385 390 395 aag gtc tac cgcacc atc gcc aac acc gac gtc gcg gct tgg ccg aac 1247 Lys Val Tyr Arg ThrIle Ala Asn Thr Asp Val Ala Ala Trp Pro Asn 400 405 410 415 ggc aag gtctac ctg ggc gtc acg aag gtc gac ttc tcc cag tac gat 1295 Gly Lys Val TyrLeu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp 420 425 430 gac cag aagaat gaa acc tcc acc cag acc tac gac tcc aag cgc aac 1343 Asp Gln Lys AsnGlu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn 435 440 445 aat ggc cacgtc tcc gcc cag gac tcc atc gac cag ctg ccg cct gag 1391 Asn Gly His ValSer Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu 450 455 460 acc act gacgag ccc ctg gag aag gcc tac tcc cac cag ctg aac tac 1439 Thr Thr Asp GluPro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr 465 470 475 gcg gag tgcttc ctg atg caa gac cgc agg ggc acc atc ccc ttc ttc 1487 Ala Glu Cys PheLeu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe 480 485 490 495 acc tggacc cac cgc tcc gtc gac ttc ttc aac acc atc gac gcc gag 1535 Thr Trp ThrHis Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala Glu 500 505 510 aag atcacc cag ctg ccc gtg gtc aag gcc tac gcc ctg tcc tcg ggt 1583 Lys Ile ThrGln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly 515 520 525 gcc tccatc att gag ggt cca ggc ttc acc ggt ggc aac ctg ctg ttc 1631 Ala Ser IleIle Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe 530 535 540 ctg aaggag tcc tcg aac tcc atc gcc aag ttc aag gtc acc ctg aac 1679 Leu Lys GluSer Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn 545 550 555 tcc gctgcc ttg ctg caa cgc tac cgc gtc cgc atc cgc tac gcc tcc 1727 Ser Ala AlaLeu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser 560 565 570 575 accacg aac ctg cgc ctg ttc gtc cag aac tcc aac aat gac ttc ctg 1775 Thr ThrAsn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu 580 585 590 gtcatc tac atc aac aag acc atg aac aag gac gat gac ctg acc tac 1823 Val IleTyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr 595 600 605 cagacc ttc gac ctc gcc acc acg aac tcc aac atg ggc ttc tcg ggc 1871 Gln ThrPhe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly 610 615 620 gacaag aat gaa ctg atc att ggt gct gag tcc ttc gtc tcc aat gaa 1919 Asp LysAsn Glu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu 625 630 635 aagatc tac atc gac aag atc gag ttc atc ccc gtc cag ctg 1961 Lys Ile Tyr IleAsp Lys Ile Glu Phe Ile Pro Val Gln Leu 640 645 650 tgataggaactctgattgaa ttc 1984 10 653 PRT Artificial Sequence Description ofArtificial Sequence non- naturally occurring amino acid sequence encodedby SEQ ID NO9 10 Met Ala Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile LysVal Thr 1 5 10 15 Pro Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr ProLeu Ala Asp 20 25 30 Asn Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys GluPhe Leu Arg 35 40 45 Met Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn SerThr Val Lys 50 55 60 Asp Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln IleLeu Gly Val 65 70 75 80 Val Gly Val Pro Phe Ala Gly Ala Leu Thr Ser PheTyr Gln Ser Phe 85 90 95 Leu Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro TrpLys Ala Phe Met 100 105 110 Ala Gln Val Glu Val Leu Ile Asp Lys Lys IleGlu Glu Tyr Ala Lys 115 120 125 Ser Lys Ala Leu Ala Glu Leu Gln Gly LeuGln Asn Asn Phe Glu Asp 130 135 140 Tyr Val Asn Ala Leu Asn Ser Trp LysLys Thr Pro Leu Ser Leu Arg 145 150 155 160 Ser Lys Arg Ser Gln Gly ArgIle Arg Glu Leu Phe Ser Gln Ala Glu 165 170 175 Ser His Phe Arg Asn SerMet Pro Ser Phe Ala Val Ser Lys Phe Glu 180 185 190 Val Leu Phe Leu ProThr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu 195 200 205 Leu Leu Lys AspAla Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser 210 215 220 Glu Asp ValAla Glu Phe Tyr Arg Arg Gln Leu Lys Leu Thr Gln Gln 225 230 235 240 TyrThr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu Asn Gly Leu 245 250 255Arg Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg 260 265270 Glu Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr 275280 285 Asp Ile Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp290 295 300 Ile Phe Thr Asp Pro Ile Phe Leu Leu Thr Thr Leu Gln Lys TyrGly 305 310 315 320 Pro Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys ProHis Leu Phe 325 330 335 Asp Tyr Leu Gln Gly Ile Glu Phe His Thr Arg LeuArg Pro Gly Tyr 340 345 350 Phe Gly Lys Asp Ser Phe Asn Tyr Trp Ser GlyAsn Tyr Val Glu Thr 355 360 365 Arg Pro Ser Ile Gly Ser Ser Lys Thr IleThr Ser Pro Phe Tyr Gly 370 375 380 Asp Lys Ser Thr Glu Pro Val Gln LysLeu Ser Phe Asp Gly Gln Lys 385 390 395 400 Val Tyr Arg Thr Ile Ala AsnThr Asp Val Ala Ala Trp Pro Asn Gly 405 410 415 Lys Val Tyr Leu Gly ValThr Lys Val Asp Phe Ser Gln Tyr Asp Asp 420 425 430 Gln Lys Asn Glu ThrSer Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn 435 440 445 Gly His Val SerAla Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr 450 455 460 Thr Asp GluPro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala 465 470 475 480 GluCys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr 485 490 495Trp Thr His Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys 500 505510 Ile Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala 515520 525 Ser Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu530 535 540 Lys Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu AsnSer 545 550 555 560 Ala Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg TyrAla Ser Thr 565 570 575 Thr Asn Leu Arg Leu Phe Val Gln Asn Ser Asn AsnAsp Phe Leu Val 580 585 590 Ile Tyr Ile Asn Lys Thr Met Asn Lys Asp AspAsp Leu Thr Tyr Gln 595 600 605 Thr Phe Asp Leu Ala Thr Thr Asn Ser AsnMet Gly Phe Ser Gly Asp 610 615 620 Lys Asn Glu Leu Ile Ile Gly Ala GluSer Phe Val Ser Asn Glu Lys 625 630 635 640 Ile Tyr Ile Asp Lys Ile GluPhe Ile Pro Val Gln Leu 645 650 11 1984 DNA Artificial SequenceDescription of Artificial Sequence non- naturally occurring nucleotidesequence encoding a Cry3Bb variant 11231mv2 amino acid sequence 11 ccatg gcc aac ccc aac aat cgc tcc gag cac gac acg atc aag gtc 47 Met AlaAsn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys Val 1 5 10 15 acc cccaac tcc gag ctc cag acc aac cac aac cag tac ccg ctg gcc 95 Thr Pro AsnSer Glu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala 20 25 30 gac aac cccaac tcc acc ctg gaa gag ctg aac tac aag gag ttc ctg 143 Asp Asn Pro AsnSer Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu 35 40 45 cgc atg acc gaggac tcc tcc acg gag gtc ctg gac aac tcc acc gtc 191 Arg Met Thr Glu AspSer Ser Thr Glu Val Leu Asp Asn Ser Thr Val 50 55 60 aag gac gcc gtc gggacc ggc atc tcc gtc gtt ggg cag atc ctg ggc 239 Lys Asp Ala Val Gly ThrGly Ile Ser Val Val Gly Gln Ile Leu Gly 65 70 75 gtc gtt ggc gtc ccc ttcgca ggt gct ctc acc tcc ttc tac cag tcc 287 Val Val Gly Val Pro Phe AlaGly Ala Leu Thr Ser Phe Tyr Gln Ser 80 85 90 95 ttc ctg aac acc atc tggccc tcc gac gcc gac ccc tgg aag gcc ttc 335 Phe Leu Asn Thr Ile Trp ProSer Asp Ala Asp Pro Trp Lys Ala Phe 100 105 110 atg gcc caa gtc gaa gtcctg atc gac aag aag atc gag gag tac gcc 383 Met Ala Gln Val Glu Val LeuIle Asp Lys Lys Ile Glu Glu Tyr Ala 115 120 125 aag tcc aag gcc ctg gccgag ctg caa ggc ctg caa aac aac ttc gag 431 Lys Ser Lys Ala Leu Ala GluLeu Gln Gly Leu Gln Asn Asn Phe Glu 130 135 140 gac tac gtc aac gcg ctgaac tcc tgg aag aag acg cct ctg tcc ctg 479 Asp Tyr Val Asn Ala Leu AsnSer Trp Lys Lys Thr Pro Leu Ser Leu 145 150 155 cgc tcc aag cgc tcc caggac cgc atc cgc gag ctg ttc tcc cag gcc 527 Arg Ser Lys Arg Ser Gln AspArg Ile Arg Glu Leu Phe Ser Gln Ala 160 165 170 175 gag tcc cac ttc cgcaac tcc atg ccg tcc ttc gcc gtc tcc aag ttc 575 Glu Ser His Phe Arg AsnSer Met Pro Ser Phe Ala Val Ser Lys Phe 180 185 190 gag gtc ctg ttc ctgccc acc tac gcc cag gct gcc aac acc cac ctc 623 Glu Val Leu Phe Leu ProThr Tyr Ala Gln Ala Ala Asn Thr His Leu 195 200 205 ctg ttg ctg aag gacgcc cag gtc ttc ggc gag gaa tgg ggc tac tcc 671 Leu Leu Leu Lys Asp AlaGln Val Phe Gly Glu Glu Trp Gly Tyr Ser 210 215 220 tcg gag gac gtc gccgag ttc tac cgt cgc cag ctg aag ctg acc caa 719 Ser Glu Asp Val Ala GluPhe Tyr Arg Arg Gln Leu Lys Leu Thr Gln 225 230 235 cag tac acc gac cactgc gtc aac tgg tac aac gtc ggc ctg aac ggc 767 Gln Tyr Thr Asp His CysVal Asn Trp Tyr Asn Val Gly Leu Asn Gly 240 245 250 255 ctg agg ggc tccacc tac gac gca tgg gtc aag ttc aac cgc ttc cgc 815 Leu Arg Gly Ser ThrTyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg 260 265 270 agg gag atg accctg acc gtc ctg gac ctg atc gtc ctg ttc ccc ttc 863 Arg Glu Met Thr LeuThr Val Leu Asp Leu Ile Val Leu Phe Pro Phe 275 280 285 tac gac atc cgcctg tac tcc aag ggc gtc aag acc gag ctg acc cgc 911 Tyr Asp Ile Arg LeuTyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg 290 295 300 gac atc ttc acggac ccc atc ttc ctg ctc acg acc ctc cag aag tac 959 Asp Ile Phe Thr AspPro Ile Phe Leu Leu Thr Thr Leu Gln Lys Tyr 305 310 315 ggt ccc acc ttcctg tcc atc gag aac tcc atc cgc aag ccc cac ctg 1007 Gly Pro Thr Phe LeuSer Ile Glu Asn Ser Ile Arg Lys Pro His Leu 320 325 330 335 ttc gac tacctc cag ggc atc gag ttc cac acg cgc ctg agg cca ggc 1055 Phe Asp Tyr LeuGln Gly Ile Glu Phe His Thr Arg Leu Arg Pro Gly 340 345 350 tac ttc ggcaag gac tcc ttc aac tac tgg tcc ggc aac tac gtc gag 1103 Tyr Phe Gly LysAsp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu 355 360 365 acc agg ccctcc atc ggc tcc tcg aag acg atc acc tcc cct ttc tac 1151 Thr Arg Pro SerIle Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr 370 375 380 ggc gac aagtcc acc gag ccc gtc cag aag ctg tcc ttc gac ggc cag 1199 Gly Asp Lys SerThr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly Gln 385 390 395 aag gtc taccgc acc atc gcc aac acc gac gtc gcg gct tgg ccg aac 1247 Lys Val Tyr ArgThr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn 400 405 410 415 ggc aaggtc tac ctg ggc gtc acg aag gtc gac ttc tcc cag tac gat 1295 Gly Lys ValTyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp 420 425 430 gac cagaag aat gaa acc tcc acc cag acc tac gac tcc aag cgc aac 1343 Asp Gln LysAsn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn 435 440 445 aat ggccac gtc tcc gcc cag gac tcc atc gac cag ctg ccg cct gag 1391 Asn Gly HisVal Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu 450 455 460 acc actgac gag ccc ctg gag aag gcc tac tcc cac cag ctg aac tac 1439 Thr Thr AspGlu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr 465 470 475 gcg gagtgc ttc ctg atg caa gac cgc agg ggc acc atc ccc ttc ttc 1487 Ala Glu CysPhe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe 480 485 490 495 acctgg acc cac cgc tcc gtc gac ttc ttc aac acc atc gac gcc gag 1535 Thr TrpThr His Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala Glu 500 505 510 aagatc acc cag ctg ccc gtg gtc aag gcc tac gcc ctg tcc tcg ggt 1583 Lys IleThr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly 515 520 525 gcctcc atc att gag ggt cca ggc ttc acc ggt ggc aac ctg ctg ttc 1631 Ala SerIle Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe 530 535 540 ctgaag gag tcc tcg aac tcc atc gcc aag ttc aag gtc acc ctg aac 1679 Leu LysGlu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn 545 550 555 tccgct gcc ttg ctg caa cgc tac cgc gtc cgc atc cgc tac gcc tcc 1727 Ser AlaAla Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser 560 565 570 575acc acg aac ctg cgc ctg ttc gtc cag aac tcc aac aat gac ttc ctg 1775 ThrThr Asn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu 580 585 590gtc atc tac atc aac aag acc atg aac aag gac gat gac ctg acc tac 1823 ValIle Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr 595 600 605cag acc ttc gac ctc gcc acc acg aac tcc aac atg ggc ttc tcg ggc 1871 GlnThr Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly 610 615 620gac aag aat gaa ctg atc att ggt gct gag tcc ttc gtc tcc aat gaa 1919 AspLys Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu 625 630 635aag atc tac atc gac aag atc gag ttc atc ccc gtc cag ctg 1961 Lys Ile TyrIle Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 640 645 650 tgataggaactctgattgaa ttc 1984 12 653 PRT Artificial Sequence Description ofArtificial Sequence non- naturally occurring amino acid sequence encodedby SEQ ID NO11 12 Met Ala Asn Pro Asn Asn Arg Ser Glu His Asp Thr IleLys Val Thr 1 5 10 15 Pro Asn Ser Glu Leu Gln Thr Asn His Asn Gln TyrPro Leu Ala Asp 20 25 30 Asn Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr LysGlu Phe Leu Arg 35 40 45 Met Thr Glu Asp Ser Ser Thr Glu Val Leu Asp AsnSer Thr Val Lys 50 55 60 Asp Ala Val Gly Thr Gly Ile Ser Val Val Gly GlnIle Leu Gly Val 65 70 75 80 Val Gly Val Pro Phe Ala Gly Ala Leu Thr SerPhe Tyr Gln Ser Phe 85 90 95 Leu Asn Thr Ile Trp Pro Ser Asp Ala Asp ProTrp Lys Ala Phe Met 100 105 110 Ala Gln Val Glu Val Leu Ile Asp Lys LysIle Glu Glu Tyr Ala Lys 115 120 125 Ser Lys Ala Leu Ala Glu Leu Gln GlyLeu Gln Asn Asn Phe Glu Asp 130 135 140 Tyr Val Asn Ala Leu Asn Ser TrpLys Lys Thr Pro Leu Ser Leu Arg 145 150 155 160 Ser Lys Arg Ser Gln AspArg Ile Arg Glu Leu Phe Ser Gln Ala Glu 165 170 175 Ser His Phe Arg AsnSer Met Pro Ser Phe Ala Val Ser Lys Phe Glu 180 185 190 Val Leu Phe LeuPro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu 195 200 205 Leu Leu LysAsp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser 210 215 220 Glu AspVal Ala Glu Phe Tyr Arg Arg Gln Leu Lys Leu Thr Gln Gln 225 230 235 240Tyr Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu Asn Gly Leu 245 250255 Arg Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg 260265 270 Glu Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr275 280 285 Asp Ile Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr ArgAsp 290 295 300 Ile Phe Thr Asp Pro Ile Phe Leu Leu Thr Thr Leu Gln LysTyr Gly 305 310 315 320 Pro Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg LysPro His Leu Phe 325 330 335 Asp Tyr Leu Gln Gly Ile Glu Phe His Thr ArgLeu Arg Pro Gly Tyr 340 345 350 Phe Gly Lys Asp Ser Phe Asn Tyr Trp SerGly Asn Tyr Val Glu Thr 355 360 365 Arg Pro Ser Ile Gly Ser Ser Lys ThrIle Thr Ser Pro Phe Tyr Gly 370 375 380 Asp Lys Ser Thr Glu Pro Val GlnLys Leu Ser Phe Asp Gly Gln Lys 385 390 395 400 Val Tyr Arg Thr Ile AlaAsn Thr Asp Val Ala Ala Trp Pro Asn Gly 405 410 415 Lys Val Tyr Leu GlyVal Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp 420 425 430 Gln Lys Asn GluThr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn 435 440 445 Gly His ValSer Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr 450 455 460 Thr AspGlu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala 465 470 475 480Glu Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr 485 490495 Trp Thr His Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys 500505 510 Ile Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala515 520 525 Ser Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu PheLeu 530 535 540 Lys Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr LeuAsn Ser 545 550 555 560 Ala Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile ArgTyr Ala Ser Thr 565 570 575 Thr Asn Leu Arg Leu Phe Val Gln Asn Ser AsnAsn Asp Phe Leu Val 580 585 590 Ile Tyr Ile Asn Lys Thr Met Asn Lys AspAsp Asp Leu Thr Tyr Gln 595 600 605 Thr Phe Asp Leu Ala Thr Thr Asn SerAsn Met Gly Phe Ser Gly Asp 610 615 620 Lys Asn Glu Leu Ile Ile Gly AlaGlu Ser Phe Val Ser Asn Glu Lys 625 630 635 640 Ile Tyr Ile Asp Lys IleGlu Phe Ile Pro Val Gln Leu 645 650 13 4149 DNA Artificial SequenceDescription of Artificial Sequence expression cassette 13 gcggccgcgttaacaagctt ctgcaggtcc gatgtgagac ttttcaacaa agggtaatat 60 ccggaaacctcctcggattc cattgcccag ctatctgtca ctttattgtg aagatagtgg 120 aaaaggaaggtggctcctac aaatgccatc attgcgataa aggaaaggcc atcgttgaag 180 atgcctctgccgacagtggt cccaaagatg gacccccacc cacgaggagc atcgtggaaa 240 aagaagacgttccaaccacg tcttcaaagc aagtggattg atgtgatggt ccgatgtgag 300 acttttcaacaaagggtaat atccggaaac ctcctcggat tccattgccc agctatctgt 360 cactttattgtgaagatagt gaaaaggaag gtggctccta caaatgccat cattgcgata 420 aaggaaaggccatcgttgaa gatgcctctg ccgacagtgg tcccaaagat ggacccccac 480 ccacgaggagcatcgtggaa aaagaagacg ttccaaccac gtcttcaaag caagtggatt 540 gatgtgatatctccactgac gtaagggatg acgcacaatc ccactatcct tcgcaagacc 600 cttcctctatataaggaagt tcatttcatt tggagaggac acgctgacaa gctgactcta 660 gcagatctaccgtcttcggt acgcgctcac tccgccctct gcctttgtta ctgccacgtt 720 tctctgaatgctctcttgtg tggtgattgc tgagagtggt ttagctggat ctagaattac 780 actctgaaatcgtgttctgc ctgtgctgat tacttgccgt cctttgtagc agcaaaatat 840 agggacatggtagtacgaaa cgaagataga acctacacag caatacgaga aatgtgtaat 900 ttggtgcttagcggtattta tttaagcaca tgttggtgtt atagggcact tggattcaga 960 agtttgctgttaatttaggc acaggcttca tactacatgg gtcaatagta tagggattca 1020 tattataggcgatactataa taatttgttc gtctgcagag cttattattt gccaaaatta 1080 gatattcctattctgttttt gtttgtgtgc tgttaaattg ttaacgcctg aaggaataaa 1140 tataaatgacgaaattttga tgtttatctc tgctccttta ttgtgaccat aagtcaagat 1200 cagatgcacttgttttaaat attgttgtct gaagaaataa gtactgacag tattttgatg 1260 cttgatctgcttgtttgttg taacaaaatt taaaaataaa gagtttcctt tttgttgctc 1320 tccttacctcctgatggtat ctagtatcta ccaactgaca ctatattgct tctctttaca 1380 tacgtatcttgctcgatgcc ttctccctag tgttgaccag tgttactcac atagtctttg 1440 ctcatttcattgtaatgcag ataccaagcg gcctctagag gatcagcatg gcgcccaccg 1500 tgatgatggcctcgtcggcc accgccgtcg ctccgttcct ggggctcaag tccaccgcca 1560 gcctccccgtcgcccgccgc tcctccagaa gcctcggcaa cgtcagcaac ggcggaagga 1620 tccggtgcatgcaggtaaca aatgcatcct agctagtagt tctttgcatt gcagcagctg 1680 cagctagcgagttagtaata ggaagggaac tgatgatcca tgcatggact gatgtgtgtt 1740 gcccatcccatcccatccca tttcccaaac gaaccgaaaa caccgtacta cgtgcaggtg 1800 tggccctacggcaacaagaa gttcgagacg ctgtcgtacc tgccgccgct gtcgaccggc 1860 gggcgcatccgctgcatgca ggcc atg gca aac cct aac aat cgt tcc gaa 1911 Met Ala Asn ProAsn Asn Arg Ser Glu 1 5 cac gac acc atc aag gtt act cca aac tct gag ttgcaa act aat cac 1959 His Asp Thr Ile Lys Val Thr Pro Asn Ser Glu Leu GlnThr Asn His 10 15 20 25 aac cag tac cca ttg gct gac aat cct aac agt actctt gag gaa ctt 2007 Asn Gln Tyr Pro Leu Ala Asp Asn Pro Asn Ser Thr LeuGlu Glu Leu 30 35 40 aac tac aag gag ttt ctc cgg atg acc gaa gat agc tccact gag gtt 2055 Asn Tyr Lys Glu Phe Leu Arg Met Thr Glu Asp Ser Ser ThrGlu Val 45 50 55 ctc gat aac tct aca gtg aag gac gct gtt gga act ggc attagc gtt 2103 Leu Asp Asn Ser Thr Val Lys Asp Ala Val Gly Thr Gly Ile SerVal 60 65 70 gtg gga cag att ctt gga gtg gtt ggt gtt cca ttc gct gga gctttg 2151 Val Gly Gln Ile Leu Gly Val Val Gly Val Pro Phe Ala Gly Ala Leu75 80 85 acc agc ttc tac cag tcc ttt ctc aac acc atc tgg cct tca gat gct2199 Thr Ser Phe Tyr Gln Ser Phe Leu Asn Thr Ile Trp Pro Ser Asp Ala 9095 100 105 gat ccc tgg aag gct ttc atg gcc caa gtg gaa gtc ttg atc gataag 2247 Asp Pro Trp Lys Ala Phe Met Ala Gln Val Glu Val Leu Ile Asp Lys110 115 120 aag atc gaa gag tat gcc aag tct aaa gcc ttg gct gag ttg caaggt 2295 Lys Ile Glu Glu Tyr Ala Lys Ser Lys Ala Leu Ala Glu Leu Gln Gly125 130 135 ttg cag aac aac ttc gag gat tac gtc aac gca ctc aac agc tggaag 2343 Leu Gln Asn Asn Phe Glu Asp Tyr Val Asn Ala Leu Asn Ser Trp Lys140 145 150 aaa act ccc ttg agt ctc agg tct aag cgt tcc cag gac cgt attcgt 2391 Lys Thr Pro Leu Ser Leu Arg Ser Lys Arg Ser Gln Asp Arg Ile Arg155 160 165 gaa ctt ttc agc caa gcc gaa tcc cac ttc aga aac tcc atg cctagc 2439 Glu Leu Phe Ser Gln Ala Glu Ser His Phe Arg Asn Ser Met Pro Ser170 175 180 185 ttt gcc gtt tct aag ttc gag gtg ctc ttc ttg cca aca tacgca caa 2487 Phe Ala Val Ser Lys Phe Glu Val Leu Phe Leu Pro Thr Tyr AlaGln 190 195 200 gct gcc aac act cat ctc ttg ctt ctc aaa gac gct cag gtgttt ggt 2535 Ala Ala Asn Thr His Leu Leu Leu Leu Lys Asp Ala Gln Val PheGly 205 210 215 gag gaa tgg ggt tac tcc agt gaa gat gtt gcc gag ttc taccgt agg 2583 Glu Glu Trp Gly Tyr Ser Ser Glu Asp Val Ala Glu Phe Tyr ArgArg 220 225 230 cag ctc aag ttg act caa cag tac aca gac cac tgc gtc aactgg tac 2631 Gln Leu Lys Leu Thr Gln Gln Tyr Thr Asp His Cys Val Asn TrpTyr 235 240 245 aac gtt ggg ctc aat ggt ctt aga gga tct acc tac gac gcatgg gtg 2679 Asn Val Gly Leu Asn Gly Leu Arg Gly Ser Thr Tyr Asp Ala TrpVal 250 255 260 265 aag ttc aac agg ttt cgt aga gag atg acc ttg act gtgctc gat ctt 2727 Lys Phe Asn Arg Phe Arg Arg Glu Met Thr Leu Thr Val LeuAsp Leu 270 275 280 atc gtt ctc ttt cca ttc tac gac att cgt ctt tac tccaaa ggc gtt 2775 Ile Val Leu Phe Pro Phe Tyr Asp Ile Arg Leu Tyr Ser LysGly Val 285 290 295 aag aca gag ctg acc aga gac atc ttc acc gat ccc atcttc cta ctt 2823 Lys Thr Glu Leu Thr Arg Asp Ile Phe Thr Asp Pro Ile PheLeu Leu 300 305 310 acg acc ctg cag aaa tac ggt cca act ttt ctc tcc attgag aac agc 2871 Thr Thr Leu Gln Lys Tyr Gly Pro Thr Phe Leu Ser Ile GluAsn Ser 315 320 325 atc agg aag cct cac ctc ttc gac tat ctg caa ggc attgag ttt cac 2919 Ile Arg Lys Pro His Leu Phe Asp Tyr Leu Gln Gly Ile GluPhe His 330 335 340 345 acc agg ttg caa cct ggt tac ttc ggt aag gat tccttc aac tac tgg 2967 Thr Arg Leu Gln Pro Gly Tyr Phe Gly Lys Asp Ser PheAsn Tyr Trp 350 355 360 agc gga aac tac gtt gaa acc aga cca tcc atc ggatct agc aag acc 3015 Ser Gly Asn Tyr Val Glu Thr Arg Pro Ser Ile Gly SerSer Lys Thr 365 370 375 atc act tct cca ttc tac ggt gac aag agc act gagcca gtg cag aag 3063 Ile Thr Ser Pro Phe Tyr Gly Asp Lys Ser Thr Glu ProVal Gln Lys 380 385 390 ttg agc ttc gat ggg cag aag gtg tat aga acc atcgcc aat acc gat 3111 Leu Ser Phe Asp Gly Gln Lys Val Tyr Arg Thr Ile AlaAsn Thr Asp 395 400 405 gtt gca gct tgg cct aat ggc aag gtc tac ctt ggagtt act aaa gtg 3159 Val Ala Ala Trp Pro Asn Gly Lys Val Tyr Leu Gly ValThr Lys Val 410 415 420 425 gac ttc tcc caa tac gac gat cag aag aac gagaca tct act caa acc 3207 Asp Phe Ser Gln Tyr Asp Asp Gln Lys Asn Glu ThrSer Thr Gln Thr 430 435 440 tac gat agt aag agg aac aat ggc cat gtt tccgca caa gac tcc att 3255 Tyr Asp Ser Lys Arg Asn Asn Gly His Val Ser AlaGln Asp Ser Ile 445 450 455 gac caa ctt cca cct gaa acc act gat gaa ccattg gag aag gct tac 3303 Asp Gln Leu Pro Pro Glu Thr Thr Asp Glu Pro LeuGlu Lys Ala Tyr 460 465 470 agt cac caa ctt aac tac gcc gaa tgc ttt ctcatg caa gac agg cgt 3351 Ser His Gln Leu Asn Tyr Ala Glu Cys Phe Leu MetGln Asp Arg Arg 475 480 485 ggc acc att ccg ttc ttt aca tgg act cac aggtct gtc gac ttc ttt 3399 Gly Thr Ile Pro Phe Phe Thr Trp Thr His Arg SerVal Asp Phe Phe 490 495 500 505 aac act atc gac gct gag aag att acc caactt ccc gtg gtc aag gct 3447 Asn Thr Ile Asp Ala Glu Lys Ile Thr Gln LeuPro Val Val Lys Ala 510 515 520 tat gcc ttg tcc agc gga gct tcc atc attgaa ggt cca ggc ttc acc 3495 Tyr Ala Leu Ser Ser Gly Ala Ser Ile Ile GluGly Pro Gly Phe Thr 525 530 535 ggt ggc aac ttg ctc ttc ctt aag gag tccagc aac tcc atc gcc aag 3543 Gly Gly Asn Leu Leu Phe Leu Lys Glu Ser SerAsn Ser Ile Ala Lys 540 545 550 ttc aaa gtg aca ctt aac tca gca gcc ttgctc caa cgt tac agg gtt 3591 Phe Lys Val Thr Leu Asn Ser Ala Ala Leu LeuGln Arg Tyr Arg Val 555 560 565 cgt atc aga tac gca agc act acc aat cttcgc ctc ttt gtc cag aac 3639 Arg Ile Arg Tyr Ala Ser Thr Thr Asn Leu ArgLeu Phe Val Gln Asn 570 575 580 585 agc aac aat gat ttc ctt gtc atc tacatc aac aag act atg aac aaa 3687 Ser Asn Asn Asp Phe Leu Val Ile Tyr IleAsn Lys Thr Met Asn Lys 590 595 600 gac gat gac ctc acc tac caa aca ttcgat ctt gcc act acc aat agt 3735 Asp Asp Asp Leu Thr Tyr Gln Thr Phe AspLeu Ala Thr Thr Asn Ser 605 610 615 aac atg gga ttc tct ggt gac aag aacgag ctg atc ata ggt gct gag 3783 Asn Met Gly Phe Ser Gly Asp Lys Asn GluLeu Ile Ile Gly Ala Glu 620 625 630 agc ttt gtc tct aat gag aag att tacata gac aag atc gag ttc att 3831 Ser Phe Val Ser Asn Glu Lys Ile Tyr IleAsp Lys Ile Glu Phe Ile 635 640 645 cca gtt caa ctc taatagatcccccgggctgc aggaattccc gatcgttcaa 3883 Pro Val Gln Leu 650 acatttggcaataaagtttc ttaagattga atcctgttgc cggtcttgcg atgattatca 3943 tataatttctgttgaattac gttaagcatg taataattaa catgtaatgc atgacgttat 4003 ttatgagatgggtttttatg attagagtcc cgcaattata catttaatac gcgatagaaa 4063 acaaaatatagcgcgcaaac taggataaat tatcgcgcgc ggtgtcatct atgttactag 4123 atcggggatatccccggggc ggccgc 4149 14 653 PRT Artificial Sequence Description ofArtificial Sequence peptide encoded by SEQ ID NO13 14 Met Ala Asn ProAsn Asn Arg Ser Glu His Asp Thr Ile Lys Val Thr 1 5 10 15 Pro Asn SerGlu Leu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp 20 25 30 Asn Pro AsnSer Thr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg 35 40 45 Met Thr GluAsp Ser Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys 50 55 60 Asp Ala ValGly Thr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val 65 70 75 80 Val GlyVal Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe 85 90 95 Leu AsnThr Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met 100 105 110 AlaGln Val Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys 115 120 125Ser Lys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp 130 135140 Tyr Val Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg 145150 155 160 Ser Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln AlaGlu 165 170 175 Ser His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser LysPhe Glu 180 185 190 Val Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn ThrHis Leu Leu 195 200 205 Leu Leu Lys Asp Ala Gln Val Phe Gly Glu Glu TrpGly Tyr Ser Ser 210 215 220 Glu Asp Val Ala Glu Phe Tyr Arg Arg Gln LeuLys Leu Thr Gln Gln 225 230 235 240 Tyr Thr Asp His Cys Val Asn Trp TyrAsn Val Gly Leu Asn Gly Leu 245 250 255 Arg Gly Ser Thr Tyr Asp Ala TrpVal Lys Phe Asn Arg Phe Arg Arg 260 265 270 Glu Met Thr Leu Thr Val LeuAsp Leu Ile Val Leu Phe Pro Phe Tyr 275 280 285 Asp Ile Arg Leu Tyr SerLys Gly Val Lys Thr Glu Leu Thr Arg Asp 290 295 300 Ile Phe Thr Asp ProIle Phe Leu Leu Thr Thr Leu Gln Lys Tyr Gly 305 310 315 320 Pro Thr PheLeu Ser Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe 325 330 335 Asp TyrLeu Gln Gly Ile Glu Phe His Thr Arg Leu Gln Pro Gly Tyr 340 345 350 PheGly Lys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr 355 360 365Arg Pro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr Gly 370 375380 Asp Lys Ser Thr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys 385390 395 400 Val Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro AsnGly 405 410 415 Lys Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln TyrAsp Asp 420 425 430 Gln Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser LysArg Asn Asn 435 440 445 Gly His Val Ser Ala Gln Asp Ser Ile Asp Gln LeuPro Pro Glu Thr 450 455 460 Thr Asp Glu Pro Leu Glu Lys Ala Tyr Ser HisGln Leu Asn Tyr Ala 465 470 475 480 Glu Cys Phe Leu Met Gln Asp Arg ArgGly Thr Ile Pro Phe Phe Thr 485 490 495 Trp Thr His Arg Ser Val Asp PhePhe Asn Thr Ile Asp Ala Glu Lys 500 505 510 Ile Thr Gln Leu Pro Val ValLys Ala Tyr Ala Leu Ser Ser Gly Ala 515 520 525 Ser Ile Ile Glu Gly ProGly Phe Thr Gly Gly Asn Leu Leu Phe Leu 530 535 540 Lys Glu Ser Ser AsnSer Ile Ala Lys Phe Lys Val Thr Leu Asn Ser 545 550 555 560 Ala Ala LeuLeu Gln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr 565 570 575 Thr AsnLeu Arg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val 580 585 590 IleTyr Ile Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr Gln 595 600 605Thr Phe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp 610 615620 Lys Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys 625630 635 640 Ile Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645 65015 3754 DNA Artificial Sequence Description of Artificial Sequenceexpression cassette 15 gcggccgcgt taacaagctt ctgcaggtcc gatgtgagacttttcaacaa agggtaatat 60 ccggaaacct cctcggattc cattgcccag ctatctgtcactttattgtg aagatagtgg 120 aaaaggaagg tggctcctac aaatgccatc attgcgataaaggaaaggcc atcgttgaag 180 atgcctctgc cgacagtggt cccaaagatg gacccccacccacgaggagc atcgtggaaa 240 aagaagacgt tccaaccacg tcttcaaagc aagtggattgatgtgatggt ccgatgtgag 300 acttttcaac aaagggtaat atccggaaac ctcctcggattccattgccc agctatctgt 360 cactttattg tgaagatagt ggaaaaggaa ggtggctcctacaaatgcca tcattgcgat 420 aaaggaaagg ccatcgttga agatgcctct gccgacagtggtcccaaaga tggaccccca 480 cccacgagga gcatcgtgga aaaagaagac gttccaaccacgtcttcaaa gcaagtggat 540 tgatgtgata tctccactga cgtaagggat gacgcacaatcccactatcc ttcgcaagac 600 ccttcctcta tataaggaag ttcatttcat ttggagaggacacgctgaca agctgactct 660 agcagatcta ccgtcttcgg tacgcgctca ctccgccctctgcctttgtt actgccacgt 720 ttctctgaat gctctcttgt gtggtgattg ctgagagtggtttagctgga tctagaatta 780 cactctgaaa tcgtgttctg cctgtgctga ttacttgccgtcctttgtag cagcaaaata 840 tagggacatg gtagtacgaa acgaagatag aacctacacagcaatacgag aaatgtgtaa 900 tttggtgctt agcggtattt atttaagcac atgttggtgttatagggcac ttggattcag 960 aagtttgctg ttaatttagg cacaggcttc atactacatgggtcaatagt atagggattc 1020 atattatagg cgatactata ataatttgtt cgtctgcagagcttattatt tgccaaaatt 1080 agatattcct attctgtttt tgtttgtgtg ctgttaaattgttaacgcct gaaggaataa 1140 atataaatga cgaaattttg atgtttatct ctgctcctttattgtgacca taagtcaaga 1200 tcagatgcac ttgttttaaa tattgttgtc tgaagaaataagtactgaca gtattttgat 1260 gcattgatct gcttgtttgt tgtaacaaaa tttaaaaataaagagtttcc tttttgttgc 1320 tctccttacc tcctgatggt atctagtatc taccaactgacactatattg cttctcttta 1380 catacgtatc ttgctcgatg ccttctccct agtgttgaccagtgttactc acatagtctt 1440 tgctcatttc attgtaatgc agataccaag cggcctctagaggatctcc atg gca aac 1498 Met Ala Asn 1 cct aac aat cgt tcc gaa cac gacacc atc aag gtt act cca aac tct 1546 Pro Asn Asn Arg Ser Glu His Asp ThrIle Lys Val Thr Pro Asn Ser 5 10 15 gag ttg caa act aat cac aac cag taccca ttg gct gac aat cct aac 1594 Glu Leu Gln Thr Asn His Asn Gln Tyr ProLeu Ala Asp Asn Pro Asn 20 25 30 35 agt act ctt gag gaa ctt aac tac aaggag ttt ctc cgg atg acc gaa 1642 Ser Thr Leu Glu Glu Leu Asn Tyr Lys GluPhe Leu Arg Met Thr Glu 40 45 50 gat agc tcc act gag gtt ctc gat aac tctaca gtg aag gac gct gtt 1690 Asp Ser Ser Thr Glu Val Leu Asp Asn Ser ThrVal Lys Asp Ala Val 55 60 65 gga act ggc att agc gtt gtg gga cag att cttgga gtg gtt ggt gtt 1738 Gly Thr Gly Ile Ser Val Val Gly Gln Ile Leu GlyVal Val Gly Val 70 75 80 cca ttc gct gga gct ttg acc agc ttc tac cag tccttt ctc aac acc 1786 Pro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser PheLeu Asn Thr 85 90 95 atc tgg cct tca gat gct gat ccc tgg aag gct ttc atggcc caa gtg 1834 Ile Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met AlaGln Val 100 105 110 115 gaa gtc ttg atc gat aag aag atc gaa gag tat gccaag tct aaa gcc 1882 Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala LysSer Lys Ala 120 125 130 ttg gct gag ttg caa ggt ttg cag aac aac ttc gaggat tac gtc aac 1930 Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu AspTyr Val Asn 135 140 145 gca ctc aac agc tgg aag aaa act ccc ttg agt ctcagg tct aag cgt 1978 Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu ArgSer Lys Arg 150 155 160 tcc cag gac cgt att cgt gaa ctt ttc agc caa gccgaa tcc cac ttc 2026 Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala GluSer His Phe 165 170 175 aga aac tcc atg cct agc ttt gcc gtt tct aag ttcgag gtg ctc ttc 2074 Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe GluVal Leu Phe 180 185 190 195 ttg cca aca tac gca caa gct gcc aac act catctc ttg ctt ctc aaa 2122 Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His LeuLeu Leu Leu Lys 200 205 210 gac gct cag gtg ttt ggt gag gaa tgg ggt tactcc agt gaa gat gtt 2170 Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr SerSer Glu Asp Val 215 220 225 gcc gag ttc tac cgt agg cag ctc aag ttg actcaa cag tac aca gac 2218 Ala Glu Phe Tyr Arg Arg Gln Leu Lys Leu Thr GlnGln Tyr Thr Asp 230 235 240 cac tgc gtc aac tgg tac aac gtt ggg ctc aatggt ctt aga gga tct 2266 His Cys Val Asn Trp Tyr Asn Val Gly Leu Asn GlyLeu Arg Gly Ser 245 250 255 acc tac gac gca tgg gtg aag ttc aac agg tttcgt aga gag atg acc 2314 Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe ArgArg Glu Met Thr 260 265 270 275 ttg act gtg ctc gat ctt atc gtt ctc tttcca ttc tac gac att cgt 2362 Leu Thr Val Leu Asp Leu Ile Val Leu Phe ProPhe Tyr Asp Ile Arg 280 285 290 ctt tac tcc aaa ggc gtt aag aca gag ctgacc aga gac atc ttc acc 2410 Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu ThrArg Asp Ile Phe Thr 295 300 305 gat ccc atc ttc cta ctt acg acc ctg cagaaa tac ggt cca act ttt 2458 Asp Pro Ile Phe Leu Leu Thr Thr Leu Gln LysTyr Gly Pro Thr Phe 310 315 320 ctc tcc att gag aac agc atc agg aag cctcac ctc ttc gac tat ctg 2506 Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro HisLeu Phe Asp Tyr Leu 325 330 335 caa ggc att gag ttt cac acc agg ttg caacct ggt tac ttc ggt aag 2554 Gln Gly Ile Glu Phe His Thr Arg Leu Gln ProGly Tyr Phe Gly Lys 340 345 350 355 gat tcc ttc aac tac tgg agc gga aactac gtt gaa acc aga cca tcc 2602 Asp Ser Phe Asn Tyr Trp Ser Gly Asn TyrVal Glu Thr Arg Pro Ser 360 365 370 atc gga tct agc aag acc atc act tctcca ttc tac ggt gac aag agc 2650 Ile Gly Ser Ser Lys Thr Ile Thr Ser ProPhe Tyr Gly Asp Lys Ser 375 380 385 act gag cca gtg cag aag ttg agc ttcgat ggg cag aag gtg tat aga 2698 Thr Glu Pro Val Gln Lys Leu Ser Phe AspGly Gln Lys Val Tyr Arg 390 395 400 acc atc gcc aat acc gat gtt gca gcttgg cct aat ggc aag gtc tac 2746 Thr Ile Ala Asn Thr Asp Val Ala Ala TrpPro Asn Gly Lys Val Tyr 405 410 415 ctt gga gtt act aaa gtg gac ttc tcccaa tac gac gat cag aag aac 2794 Leu Gly Val Thr Lys Val Asp Phe Ser GlnTyr Asp Asp Gln Lys Asn 420 425 430 435 gag aca tct act caa acc tac gatagt aag agg aac aat ggc cat gtt 2842 Glu Thr Ser Thr Gln Thr Tyr Asp SerLys Arg Asn Asn Gly His Val 440 445 450 tcc gca caa gac tcc att gac caactt cca cct gaa acc act gat gaa 2890 Ser Ala Gln Asp Ser Ile Asp Gln LeuPro Pro Glu Thr Thr Asp Glu 455 460 465 cca ttg gag aag gct tac agt caccaa ctt aac tac gcc gaa tgc ttt 2938 Pro Leu Glu Lys Ala Tyr Ser His GlnLeu Asn Tyr Ala Glu Cys Phe 470 475 480 ctc atg caa gac agg cgt ggc accatt ccg ttc ttt aca tgg act cac 2986 Leu Met Gln Asp Arg Arg Gly Thr IlePro Phe Phe Thr Trp Thr His 485 490 495 agg tct gtc gac ttc ttt aac actatc gac gct gag aag att acc caa 3034 Arg Ser Val Asp Phe Phe Asn Thr IleAsp Ala Glu Lys Ile Thr Gln 500 505 510 515 ctt ccc gtg gtc aag gct tatgcc ttg tcc agc gga gct tcc atc att 3082 Leu Pro Val Val Lys Ala Tyr AlaLeu Ser Ser Gly Ala Ser Ile Ile 520 525 530 gaa ggt cca ggc ttc acc ggtggc aac ttg ctc ttc ctt aag gag tcc 3130 Glu Gly Pro Gly Phe Thr Gly GlyAsn Leu Leu Phe Leu Lys Glu Ser 535 540 545 agc aac tcc atc gcc aag ttcaaa gtg aca ctt aac tca gca gcc ttg 3178 Ser Asn Ser Ile Ala Lys Phe LysVal Thr Leu Asn Ser Ala Ala Leu 550 555 560 ctc caa cgt tac agg gtt cgtatc aga tac gca agc act acc aat ctt 3226 Leu Gln Arg Tyr Arg Val Arg IleArg Tyr Ala Ser Thr Thr Asn Leu 565 570 575 cgc ctc ttt gtc cag aac agcaac aat gat ttc ctt gtc atc tac atc 3274 Arg Leu Phe Val Gln Asn Ser AsnAsn Asp Phe Leu Val Ile Tyr Ile 580 585 590 595 aac aag act atg aac aaagac gat gac ctc acc tac caa aca ttc gat 3322 Asn Lys Thr Met Asn Lys AspAsp Asp Leu Thr Tyr Gln Thr Phe Asp 600 605 610 ctt gcc act acc aat agtaac atg gga ttc tct ggt gac aag aac gag 3370 Leu Ala Thr Thr Asn Ser AsnMet Gly Phe Ser Gly Asp Lys Asn Glu 615 620 625 ctg atc ata ggt gct gagagc ttt gtc tct aat gag aag att tac ata 3418 Leu Ile Ile Gly Ala Glu SerPhe Val Ser Asn Glu Lys Ile Tyr Ile 630 635 640 gac aag atc gag ttc attcca gtt caa ctc taatagatcc cccgggctgc 3468 Asp Lys Ile Glu Phe Ile ProVal Gln Leu 645 650 aggaattccc gatcgttcaa acatttggca ataaagtttcttaagattga atcctgttgc 3528 cggtcttgcg atgattatca tataatttct gttgaattacgttaagcatg taataattaa 3588 catgtaatgc atgacgttat ttatgagatg ggtttttatgattagagtcc cgcaattata 3648 catttaatac gcgatagaaa acaaaatata gcgcgcaaactaggataaat tatcgcgcgc 3708 ggtgtcatct atgttactag atcggggata tccccggggcggccgc 3754 16 653 PRT Artificial Sequence PRT (1)..(653) Cry3Bb1variant v11231 16 Met Ala Asn Pro Asn Asn Arg Ser Glu His Asp Thr IleLys Val Thr 1 5 10 15 Pro Asn Ser Glu Leu Gln Thr Asn His Asn Gln TyrPro Leu Ala Asp 20 25 30 Asn Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr LysGlu Phe Leu Arg 35 40 45 Met Thr Glu Asp Ser Ser Thr Glu Val Leu Asp AsnSer Thr Val Lys 50 55 60 Asp Ala Val Gly Thr Gly Ile Ser Val Val Gly GlnIle Leu Gly Val 65 70 75 80 Val Gly Val Pro Phe Ala Gly Ala Leu Thr SerPhe Tyr Gln Ser Phe 85 90 95 Leu Asn Thr Ile Trp Pro Ser Asp Ala Asp ProTrp Lys Ala Phe Met 100 105 110 Ala Gln Val Glu Val Leu Ile Asp Lys LysIle Glu Glu Tyr Ala Lys 115 120 125 Ser Lys Ala Leu Ala Glu Leu Gln GlyLeu Gln Asn Asn Phe Glu Asp 130 135 140 Tyr Val Asn Ala Leu Asn Ser TrpLys Lys Thr Pro Leu Ser Leu Arg 145 150 155 160 Ser Lys Arg Ser Gln AspArg Ile Arg Glu Leu Phe Ser Gln Ala Glu 165 170 175 Ser His Phe Arg AsnSer Met Pro Ser Phe Ala Val Ser Lys Phe Glu 180 185 190 Val Leu Phe LeuPro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu 195 200 205 Leu Leu LysAsp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser 210 215 220 Glu AspVal Ala Glu Phe Tyr Arg Arg Gln Leu Lys Leu Thr Gln Gln 225 230 235 240Tyr Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu Asn Gly Leu 245 250255 Arg Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg 260265 270 Glu Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr275 280 285 Asp Ile Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr ArgAsp 290 295 300 Ile Phe Thr Asp Pro Ile Phe Leu Leu Thr Thr Leu Gln LysTyr Gly 305 310 315 320 Pro Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg LysPro His Leu Phe 325 330 335 Asp Tyr Leu Gln Gly Ile Glu Phe His Thr ArgLeu Gln Pro Gly Tyr 340 345 350 Phe Gly Lys Asp Ser Phe Asn Tyr Trp SerGly Asn Tyr Val Glu Thr 355 360 365 Arg Pro Ser Ile Gly Ser Ser Lys ThrIle Thr Ser Pro Phe Tyr Gly 370 375 380 Asp Lys Ser Thr Glu Pro Val GlnLys Leu Ser Phe Asp Gly Gln Lys 385 390 395 400 Val Tyr Arg Thr Ile AlaAsn Thr Asp Val Ala Ala Trp Pro Asn Gly 405 410 415 Lys Val Tyr Leu GlyVal Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp 420 425 430 Gln Lys Asn GluThr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn 435 440 445 Gly His ValSer Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr 450 455 460 Thr AspGlu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala 465 470 475 480Glu Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr 485 490495 Trp Thr His Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys 500505 510 Ile Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala515 520 525 Ser Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu PheLeu 530 535 540 Lys Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr LeuAsn Ser 545 550 555 560 Ala Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile ArgTyr Ala Ser Thr 565 570 575 Thr Asn Leu Arg Leu Phe Val Gln Asn Ser AsnAsn Asp Phe Leu Val 580 585 590 Ile Tyr Ile Asn Lys Thr Met Asn Lys AspAsp Asp Leu Thr Tyr Gln 595 600 605 Thr Phe Asp Leu Ala Thr Thr Asn SerAsn Met Gly Phe Ser Gly Asp 610 615 620 Lys Asn Glu Leu Ile Ile Gly AlaGlu Ser Phe Val Ser Asn Glu Lys 625 630 635 640 Ile Tyr Ile Asp Lys IleGlu Phe Ile Pro Val Gln Leu 645 650 17 3450 DNA Artificial SequenceDescription of Artificial Sequence expression cassette 17 gcggccgcgttaacaagctt ctgacgtaag ggatgacgca cctgacgtaa gggatgacgc 60 acctgacgtaagggatgacg cacctgacgt aagggatgac gcactcgaga tccccatctc 120 cactgacgtaagggatgacg cacaatccca ctatccttcg caagaccctt cctctatata 180 aggaagttcatttcatttgg agaggacacg ctgacaagct agcttggctg caggtagatc 240 ctagaaccatcttccacaca ctcaagccac actattggag aacacacagg gacaacacac 300 cataagatccaagggaggcc tccgccgccg ccggtaacca ccccgcccct ctcctctttc 360 tttctccgtttttttttccg tctcggtctc gatctttggc cttggtagtt tgggtgggcg 420 agaggcggcttcgtgcgcgc ccagatcggt gcgcgggagg ggcgggatct cgcggctggg 480 gctctcgccggcgtggatcc ggcccggatc tcgcggggaa tggggctctc ggatgtagat 540 ctgcgatccgccgttgttgg gggagatgat ggggggttta aaatttccgc cgtgctaaac 600 aagatcaggaagaggggaaa agggcactat ggtttatatt tttatatatt tctgctgctt 660 cgtcaggcttagatgtgcta gatctttctt tcttcttttt gtgggtagaa tttgaatccc 720 tcagcattgttcatcggtag tttttctttt catgatttgt gacaaatgca gcctcgtgcg 780 gagcttttttgtaggtagaa gtgatcaacc tctagaggat cagcatggcg cccaccgtga 840 tgatggcctcgtcggccacc gccgtcgctc cgttcctggg gctcaagtcc accgccagcc 900 tccccgtcgcccgccgctcc tccagaagcc tcggcaacgt cagcaacggc ggaaggatcc 960 ggtgcatgcaggtaacaaat gcatcctagc tagtagttct ttgcattgca gcagctgcag 1020 ctagcgagttagtaatagga agggaactga tgatccatgc atggactgat gtgtgttgcc 1080 catcccatcccatcccattt cccaaacgaa ccgaaaacac cgtactacgt gcaggtgtgg 1140 ccctacggcaacaagaagtt cgagacgctg tcgtacctgc cgccgctgtc gaccggcggg 1200 cgcatccgctgcatgcaggc c atg gcc aac ccc aac aat cgc tcc gag cac 1251 Met Ala AsnPro Asn Asn Arg Ser Glu His 1 5 10 gac acg atc aag gtc acc ccc aac tccgag ctc cag acc aac cac aac 1299 Asp Thr Ile Lys Val Thr Pro Asn Ser GluLeu Gln Thr Asn His Asn 15 20 25 cag tac ccg ctg gcc gac aac ccc aac tccacc ctg gaa gag ctg aac 1347 Gln Tyr Pro Leu Ala Asp Asn Pro Asn Ser ThrLeu Glu Glu Leu Asn 30 35 40 tac aag gag ttc ctg cgc atg acc gag gac tcctcc acg gag gtc ctg 1395 Tyr Lys Glu Phe Leu Arg Met Thr Glu Asp Ser SerThr Glu Val Leu 45 50 55 gac aac tcc acc gtc aag gac gcc gtc ggg acc ggcatc tcc gtc gtt 1443 Asp Asn Ser Thr Val Lys Asp Ala Val Gly Thr Gly IleSer Val Val 60 65 70 ggg cag atc ctg ggc gtc gtt ggc gtc ccc ttc gca ggtgct ctc acc 1491 Gly Gln Ile Leu Gly Val Val Gly Val Pro Phe Ala Gly AlaLeu Thr 75 80 85 90 tcc ttc tac cag tcc ttc ctg aac acc atc tgg ccc tccgac gcc gac 1539 Ser Phe Tyr Gln Ser Phe Leu Asn Thr Ile Trp Pro Ser AspAla Asp 95 100 105 ccc tgg aag gcc ttc atg gcc caa gtc gaa gtc ctg atcgac aag aag 1587 Pro Trp Lys Ala Phe Met Ala Gln Val Glu Val Leu Ile AspLys Lys 110 115 120 atc gag gag tac gcc aag tcc aag gcc ctg gcc gag ctgcaa ggc ctg 1635 Ile Glu Glu Tyr Ala Lys Ser Lys Ala Leu Ala Glu Leu GlnGly Leu 125 130 135 caa aac aac ttc gag gac tac gtc aac gcg ctg aac tcctgg aag aag 1683 Gln Asn Asn Phe Glu Asp Tyr Val Asn Ala Leu Asn Ser TrpLys Lys 140 145 150 acg cct ctg tcc ctg cgc tcc aag cgc tcc cag ggc cgcatc cgc gag 1731 Thr Pro Leu Ser Leu Arg Ser Lys Arg Ser Gln Gly Arg IleArg Glu 155 160 165 170 ctg ttc tcc cag gcc gag tcc cac ttc cgc aac tccatg ccg tcc ttc 1779 Leu Phe Ser Gln Ala Glu Ser His Phe Arg Asn Ser MetPro Ser Phe 175 180 185 gcc gtc tcc aag ttc gag gtc ctg ttc ctg ccc acctac gcc cag gct 1827 Ala Val Ser Lys Phe Glu Val Leu Phe Leu Pro Thr TyrAla Gln Ala 190 195 200 gcc aac acc cac ctc ctg ttg ctg aag gac gcc caggtc ttc ggc gag 1875 Ala Asn Thr His Leu Leu Leu Leu Lys Asp Ala Gln ValPhe Gly Glu 205 210 215 gaa tgg ggc tac tcc tcg gag gac gtc gcc gag ttctac cgt cgc cag 1923 Glu Trp Gly Tyr Ser Ser Glu Asp Val Ala Glu Phe TyrArg Arg Gln 220 225 230 ctg aag ctg acc caa cag tac acc gac cac tgc gtcaac tgg tac aac 1971 Leu Lys Leu Thr Gln Gln Tyr Thr Asp His Cys Val AsnTrp Tyr Asn 235 240 245 250 gtc ggc ctg aac ggc ctg agg ggc tcc acc tacgac gca tgg gtc aag 2019 Val Gly Leu Asn Gly Leu Arg Gly Ser Thr Tyr AspAla Trp Val Lys 255 260 265 ttc aac cgc ttc cgc agg gag atg acc ctg accgtc ctg gac ctg atc 2067 Phe Asn Arg Phe Arg Arg Glu Met Thr Leu Thr ValLeu Asp Leu Ile 270 275 280 gtc ctg ttc ccc ttc tac gac atc cgc ctg tactcc aag ggc gtc aag 2115 Val Leu Phe Pro Phe Tyr Asp Ile Arg Leu Tyr SerLys Gly Val Lys 285 290 295 acc gag ctg acc cgc gac atc ttc acg gac cccatc ttc ctg ctc acg 2163 Thr Glu Leu Thr Arg Asp Ile Phe Thr Asp Pro IlePhe Leu Leu Thr 300 305 310 acc ctc cag aag tac ggt ccc acc ttc ctg tccatc gag aac tcc atc 2211 Thr Leu Gln Lys Tyr Gly Pro Thr Phe Leu Ser IleGlu Asn Ser Ile 315 320 325 330 cgc aag ccc cac ctg ttc gac tac ctc cagggc atc gag ttc cac acg 2259 Arg Lys Pro His Leu Phe Asp Tyr Leu Gln GlyIle Glu Phe His Thr 335 340 345 cgc ctg agg cca ggc tac ttc ggc aag gactcc ttc aac tac tgg tcc 2307 Arg Leu Arg Pro Gly Tyr Phe Gly Lys Asp SerPhe Asn Tyr Trp Ser 350 355 360 ggc aac tac gtc gag acc agg ccc tcc atcggc tcc tcg aag acg atc 2355 Gly Asn Tyr Val Glu Thr Arg Pro Ser Ile GlySer Ser Lys Thr Ile 365 370 375 acc tcc cct ttc tac ggc gac aag tcc accgag ccc gtc cag aag ctg 2403 Thr Ser Pro Phe Tyr Gly Asp Lys Ser Thr GluPro Val Gln Lys Leu 380 385 390 tcc ttc gac ggc cag aag gtc tac cgc accatc gcc aac acc gac gtc 2451 Ser Phe Asp Gly Gln Lys Val Tyr Arg Thr IleAla Asn Thr Asp Val 395 400 405 410 gcg gct tgg ccg aac ggc aag gtc tacctg ggc gtc acg aag gtc gac 2499 Ala Ala Trp Pro Asn Gly Lys Val Tyr LeuGly Val Thr Lys Val Asp 415 420 425 ttc tcc cag tac gat gac cag aag aatgaa acc tcc acc cag acc tac 2547 Phe Ser Gln Tyr Asp Asp Gln Lys Asn GluThr Ser Thr Gln Thr Tyr 430 435 440 gac tcc aag cgc aac aat ggc cac gtctcc gcc cag gac tcc atc gac 2595 Asp Ser Lys Arg Asn Asn Gly His Val SerAla Gln Asp Ser Ile Asp 445 450 455 cag ctg ccg cct gag acc act gac gagccc ctg gag aag gcc tac tcc 2643 Gln Leu Pro Pro Glu Thr Thr Asp Glu ProLeu Glu Lys Ala Tyr Ser 460 465 470 cac cag ctg aac tac gcg gag tgc ttcctg atg caa gac cgc agg ggc 2691 His Gln Leu Asn Tyr Ala Glu Cys Phe LeuMet Gln Asp Arg Arg Gly 475 480 485 490 acc atc ccc ttc ttc acc tgg acccac cgc tcc gtc gac ttc ttc aac 2739 Thr Ile Pro Phe Phe Thr Trp Thr HisArg Ser Val Asp Phe Phe Asn 495 500 505 acc atc gac gcc gag aag atc acccag ctg ccc gtg gtc aag gcc tac 2787 Thr Ile Asp Ala Glu Lys Ile Thr GlnLeu Pro Val Val Lys Ala Tyr 510 515 520 gcc ctg tcc tcg ggt gcc tcc atcatt gag ggt cca ggc ttc acc ggt 2835 Ala Leu Ser Ser Gly Ala Ser Ile IleGlu Gly Pro Gly Phe Thr Gly 525 530 535 ggc aac ctg ctg ttc ctg aag gagtcc tcg aac tcc atc gcc aag ttc 2883 Gly Asn Leu Leu Phe Leu Lys Glu SerSer Asn Ser Ile Ala Lys Phe 540 545 550 aag gtc acc ctg aac tcc gct gccttg ctg caa cgc tac cgc gtc cgc 2931 Lys Val Thr Leu Asn Ser Ala Ala LeuLeu Gln Arg Tyr Arg Val Arg 555 560 565 570 atc cgc tac gcc tcc acc acgaac ctg cgc ctg ttc gtc cag aac tcc 2979 Ile Arg Tyr Ala Ser Thr Thr AsnLeu Arg Leu Phe Val Gln Asn Ser 575 580 585 aac aat gac ttc ctg gtc atctac atc aac aag acc atg aac aag gac 3027 Asn Asn Asp Phe Leu Val Ile TyrIle Asn Lys Thr Met Asn Lys Asp 590 595 600 gat gac ctg acc tac cag accttc gac ctc gcc acc acg aac tcc aac 3075 Asp Asp Leu Thr Tyr Gln Thr PheAsp Leu Ala Thr Thr Asn Ser Asn 605 610 615 atg ggc ttc tcg ggc gac aagaat gaa ctg atc att ggt gct gag tcc 3123 Met Gly Phe Ser Gly Asp Lys AsnGlu Leu Ile Ile Gly Ala Glu Ser 620 625 630 ttc gtc tcc aat gaa aag atctac atc gac aag atc gag ttc atc ccc 3171 Phe Val Ser Asn Glu Lys Ile TyrIle Asp Lys Ile Glu Phe Ile Pro 635 640 645 650 gtc cag ctg tgataggaactctgattgaa ttctgcatgc gtttggacgt 3220 Val Gln Leu atgctcattc aggttggagccaatttggtt gatgtgtgtg cgagttcttg cgagtctgat 3280 gagacatctc tgtattgtgtttctttcccc agtgttttct gtacttgtgt aatcggctaa 3340 tcgccaacag attcggcgatgaataaatga gaaataaatt gttctgattt tgagtgcaaa 3400 aaaaaaggaa ttagatctgtgtgtgttttt tggatccccg gggcggccgc 3450 18 653 PRT Artificial Sequence PRT(1)..(653) Cry3Bb1 variant 11231mv1 18 Met Ala Asn Pro Asn Asn Arg SerGlu His Asp Thr Ile Lys Val Thr 1 5 10 15 Pro Asn Ser Glu Leu Gln ThrAsn His Asn Gln Tyr Pro Leu Ala Asp 20 25 30 Asn Pro Asn Ser Thr Leu GluGlu Leu Asn Tyr Lys Glu Phe Leu Arg 35 40 45 Met Thr Glu Asp Ser Ser ThrGlu Val Leu Asp Asn Ser Thr Val Lys 50 55 60 Asp Ala Val Gly Thr Gly IleSer Val Val Gly Gln Ile Leu Gly Val 65 70 75 80 Val Gly Val Pro Phe AlaGly Ala Leu Thr Ser Phe Tyr Gln Ser Phe 85 90 95 Leu Asn Thr Ile Trp ProSer Asp Ala Asp Pro Trp Lys Ala Phe Met 100 105 110 Ala Gln Val Glu ValLeu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys 115 120 125 Ser Lys Ala LeuAla Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp 130 135 140 Tyr Val AsnAla Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg 145 150 155 160 SerLys Arg Ser Gln Gly Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu 165 170 175Ser His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu 180 185190 Val Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu 195200 205 Leu Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser210 215 220 Glu Asp Val Ala Glu Phe Tyr Arg Arg Gln Leu Lys Leu Thr GlnGln 225 230 235 240 Tyr Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly LeuAsn Gly Leu 245 250 255 Arg Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe AsnArg Phe Arg Arg 260 265 270 Glu Met Thr Leu Thr Val Leu Asp Leu Ile ValLeu Phe Pro Phe Tyr 275 280 285 Asp Ile Arg Leu Tyr Ser Lys Gly Val LysThr Glu Leu Thr Arg Asp 290 295 300 Ile Phe Thr Asp Pro Ile Phe Leu LeuThr Thr Leu Gln Lys Tyr Gly 305 310 315 320 Pro Thr Phe Leu Ser Ile GluAsn Ser Ile Arg Lys Pro His Leu Phe 325 330 335 Asp Tyr Leu Gln Gly IleGlu Phe His Thr Arg Leu Arg Pro Gly Tyr 340 345 350 Phe Gly Lys Asp SerPhe Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr 355 360 365 Arg Pro Ser IleGly Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr Gly 370 375 380 Asp Lys SerThr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys 385 390 395 400 ValTyr Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly 405 410 415Lys Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp 420 425430 Gln Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn 435440 445 Gly His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr450 455 460 Thr Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn TyrAla 465 470 475 480 Glu Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile ProPhe Phe Thr 485 490 495 Trp Thr His Arg Ser Val Asp Phe Phe Asn Thr IleAsp Ala Glu Lys 500 505 510 Ile Thr Gln Leu Pro Val Val Lys Ala Tyr AlaLeu Ser Ser Gly Ala 515 520 525 Ser Ile Ile Glu Gly Pro Gly Phe Thr GlyGly Asn Leu Leu Phe Leu 530 535 540 Lys Glu Ser Ser Asn Ser Ile Ala LysPhe Lys Val Thr Leu Asn Ser 545 550 555 560 Ala Ala Leu Leu Gln Arg TyrArg Val Arg Ile Arg Tyr Ala Ser Thr 565 570 575 Thr Asn Leu Arg Leu PheVal Gln Asn Ser Asn Asn Asp Phe Leu Val 580 585 590 Ile Tyr Ile Asn LysThr Met Asn Lys Asp Asp Asp Leu Thr Tyr Gln 595 600 605 Thr Phe Asp LeuAla Thr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp 610 615 620 Lys Asn GluLeu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys 625 630 635 640 IleTyr Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645 650 19 3039 DNAArtificial Sequence Description of Artificial Sequence expressioncassette 19 gcggccgcgt taacaagctt ctgacgtaag ggatgacgca cctgacgtaagggatgacgc 60 acctgacgta agggatgacg cacctgacgt aagggatgac gcactcgagatccccatctc 120 cactgacgta agggatgacg cacaatccca ctatccttcg caagacccttcctctatata 180 aggaagttca tttcatttgg agaggacacg ctgacaagct agcttggctgcaggtagatc 240 ctagaaccat cttccacaca ctcaagccac actattggag aacacacagggacaacacac 300 cataagatcc aagggaggcc tccgccgccg ccggtaacca ccccgcccctctcctctttc 360 tttctccgtt tttttttccg tctcggtctc gatctttggc cttggtagtttgggtgggcg 420 agaggcggct tcgtgcgcgc ccagatcggt gcgcgggagg ggcgggatctcgcggctggg 480 gctctcgccg gcgtggatcc ggcccggatc tcgcggggaa tggggctctcggatgtagat 540 ctgcgatccg ccgttgttgg gggagatgat ggggggttta aaatttccgccgtgctaaac 600 aagatcagga agaggggaaa agggcactat ggtttatatt tttatatatttctgctgctt 660 cgtcaggctt agatgtgcta gatctttctt tcttcttttt gtgggtagaatttgaatccc 720 tcagcattgt tcatcggtag tttttctttt catgatttgt gacaaatgcagcctcgtgcg 780 gagctttttt gtaggtagaa gtgatcaacc atg gcc aac ccc aac aatcgc tcc 834 Met Ala Asn Pro Asn Asn Arg Ser 1 5 gag cac gac acg atc aaggtc acc ccc aac tcc gag ctc cag acc aac 882 Glu His Asp Thr Ile Lys ValThr Pro Asn Ser Glu Leu Gln Thr Asn 10 15 20 cac aac cag tac ccg ctg gccgac aac ccc aac tcc acc ctg gaa gag 930 His Asn Gln Tyr Pro Leu Ala AspAsn Pro Asn Ser Thr Leu Glu Glu 25 30 35 40 ctg aac tac aag gag ttc ctgcgc atg acc gag gac tcc tcc acg gag 978 Leu Asn Tyr Lys Glu Phe Leu ArgMet Thr Glu Asp Ser Ser Thr Glu 45 50 55 gtc ctg gac aac tcc acc gtc aaggac gcc gtc ggg acc ggc atc tcc 1026 Val Leu Asp Asn Ser Thr Val Lys AspAla Val Gly Thr Gly Ile Ser 60 65 70 gtc gtt ggg cag atc ctg ggc gtc gttggc gtc ccc ttc gca ggt gct 1074 Val Val Gly Gln Ile Leu Gly Val Val GlyVal Pro Phe Ala Gly Ala 75 80 85 ctc acc tcc ttc tac cag tcc ttc ctg aacacc atc tgg ccc tcc gac 1122 Leu Thr Ser Phe Tyr Gln Ser Phe Leu Asn ThrIle Trp Pro Ser Asp 90 95 100 gcc gac ccc tgg aag gcc ttc atg gcc caagtc gaa gtc ctg atc gac 1170 Ala Asp Pro Trp Lys Ala Phe Met Ala Gln ValGlu Val Leu Ile Asp 105 110 115 120 aag aag atc gag gag tac gcc aag tccaag gcc ctg gcc gag ctg caa 1218 Lys Lys Ile Glu Glu Tyr Ala Lys Ser LysAla Leu Ala Glu Leu Gln 125 130 135 ggc ctg caa aac aac ttc gag gac tacgtc aac gcg ctg aac tcc tgg 1266 Gly Leu Gln Asn Asn Phe Glu Asp Tyr ValAsn Ala Leu Asn Ser Trp 140 145 150 aag aag acg cct ctg tcc ctg cgc tccaag cgc tcc cag ggc cgc atc 1314 Lys Lys Thr Pro Leu Ser Leu Arg Ser LysArg Ser Gln Gly Arg Ile 155 160 165 cgc gag ctg ttc tcc cag gcc gag tcccac ttc cgc aac tcc atg ccg 1362 Arg Glu Leu Phe Ser Gln Ala Glu Ser HisPhe Arg Asn Ser Met Pro 170 175 180 tcc ttc gcc gtc tcc aag ttc gag gtcctg ttc ctg ccc acc tac gcc 1410 Ser Phe Ala Val Ser Lys Phe Glu Val LeuPhe Leu Pro Thr Tyr Ala 185 190 195 200 cag gct gcc aac acc cac ctc ctgttg ctg aag gac gcc cag gtc ttc 1458 Gln Ala Ala Asn Thr His Leu Leu LeuLeu Lys Asp Ala Gln Val Phe 205 210 215 ggc gag gaa tgg ggc tac tcc tcggag gac gtc gcc gag ttc tac cgt 1506 Gly Glu Glu Trp Gly Tyr Ser Ser GluAsp Val Ala Glu Phe Tyr Arg 220 225 230 cgc cag ctg aag ctg acc caa cagtac acc gac cac tgc gtc aac tgg 1554 Arg Gln Leu Lys Leu Thr Gln Gln TyrThr Asp His Cys Val Asn Trp 235 240 245 tac aac gtc ggc ctg aac ggc ctgagg ggc tcc acc tac gac gca tgg 1602 Tyr Asn Val Gly Leu Asn Gly Leu ArgGly Ser Thr Tyr Asp Ala Trp 250 255 260 gtc aag ttc aac cgc ttc cgc agggag atg acc ctg acc gtc ctg gac 1650 Val Lys Phe Asn Arg Phe Arg Arg GluMet Thr Leu Thr Val Leu Asp 265 270 275 280 ctg atc gtc ctg ttc ccc ttctac gac atc cgc ctg tac tcc aag ggc 1698 Leu Ile Val Leu Phe Pro Phe TyrAsp Ile Arg Leu Tyr Ser Lys Gly 285 290 295 gtc aag acc gag ctg acc cgcgac atc ttc acg gac ccc atc ttc ctg 1746 Val Lys Thr Glu Leu Thr Arg AspIle Phe Thr Asp Pro Ile Phe Leu 300 305 310 ctc acg acc ctc cag aag tacggt ccc acc ttc ctg tcc atc gag aac 1794 Leu Thr Thr Leu Gln Lys Tyr GlyPro Thr Phe Leu Ser Ile Glu Asn 315 320 325 tcc atc cgc aag ccc cac ctgttc gac tac ctc cag ggc atc gag ttc 1842 Ser Ile Arg Lys Pro His Leu PheAsp Tyr Leu Gln Gly Ile Glu Phe 330 335 340 cac acg cgc ctg agg cca ggctac ttc ggc aag gac tcc ttc aac tac 1890 His Thr Arg Leu Arg Pro Gly TyrPhe Gly Lys Asp Ser Phe Asn Tyr 345 350 355 360 tgg tcc ggc aac tac gtcgag acc agg ccc tcc atc ggc tcc tcg aag 1938 Trp Ser Gly Asn Tyr Val GluThr Arg Pro Ser Ile Gly Ser Ser Lys 365 370 375 acg atc acc tcc cct ttctac ggc gac aag tcc acc gag ccc gtc cag 1986 Thr Ile Thr Ser Pro Phe TyrGly Asp Lys Ser Thr Glu Pro Val Gln 380 385 390 aag ctg tcc ttc gac ggccag aag gtc tac cgc acc atc gcc aac acc 2034 Lys Leu Ser Phe Asp Gly GlnLys Val Tyr Arg Thr Ile Ala Asn Thr 395 400 405 gac gtc gcg gct tgg ccgaac ggc aag gtc tac ctg ggc gtc acg aag 2082 Asp Val Ala Ala Trp Pro AsnGly Lys Val Tyr Leu Gly Val Thr Lys 410 415 420 gtc gac ttc tcc cag tacgat gac cag aag aat gaa acc tcc acc cag 2130 Val Asp Phe Ser Gln Tyr AspAsp Gln Lys Asn Glu Thr Ser Thr Gln 425 430 435 440 acc tac gac tcc aagcgc aac aat ggc cac gtc tcc gcc cag gac tcc 2178 Thr Tyr Asp Ser Lys ArgAsn Asn Gly His Val Ser Ala Gln Asp Ser 445 450 455 atc gac cag ctg ccgcct gag acc act gac gag ccc ctg gag aag gcc 2226 Ile Asp Gln Leu Pro ProGlu Thr Thr Asp Glu Pro Leu Glu Lys Ala 460 465 470 tac tcc cac cag ctgaac tac gcg gag tgc ttc ctg atg caa gac cgc 2274 Tyr Ser His Gln Leu AsnTyr Ala Glu Cys Phe Leu Met Gln Asp Arg 475 480 485 agg ggc acc atc cccttc ttc acc tgg acc cac cgc tcc gtc gac ttc 2322 Arg Gly Thr Ile Pro PhePhe Thr Trp Thr His Arg Ser Val Asp Phe 490 495 500 ttc aac acc atc gacgcc gag aag atc acc cag ctg ccc gtg gtc aag 2370 Phe Asn Thr Ile Asp AlaGlu Lys Ile Thr Gln Leu Pro Val Val Lys 505 510 515 520 gcc tac gcc ctgtcc tcg ggt gcc tcc atc att gag ggt cca ggc ttc 2418 Ala Tyr Ala Leu SerSer Gly Ala Ser Ile Ile Glu Gly Pro Gly Phe 525 530 535 acc ggt ggc aacctg ctg ttc ctg aag gag tcc tcg aac tcc atc gcc 2466 Thr Gly Gly Asn LeuLeu Phe Leu Lys Glu Ser Ser Asn Ser Ile Ala 540 545 550 aag ttc aag gtcacc ctg aac tcc gct gcc ttg ctg caa cgc tac cgc 2514 Lys Phe Lys Val ThrLeu Asn Ser Ala Ala Leu Leu Gln Arg Tyr Arg 555 560 565 gtc cgc atc cgctac gcc tcc acc acg aac ctg cgc ctg ttc gtc cag 2562 Val Arg Ile Arg TyrAla Ser Thr Thr Asn Leu Arg Leu Phe Val Gln 570 575 580 aac tcc aac aatgac ttc ctg gtc atc tac atc aac aag acc atg aac 2610 Asn Ser Asn Asn AspPhe Leu Val Ile Tyr Ile Asn Lys Thr Met Asn 585 590 595 600 aag gac gatgac ctg acc tac cag acc ttc gac ctc gcc acc acg aac 2658 Lys Asp Asp AspLeu Thr Tyr Gln Thr Phe Asp Leu Ala Thr Thr Asn 605 610 615 tcc aac atgggc ttc tcg ggc gac aag aat gaa ctg atc att ggt gct 2706 Ser Asn Met GlyPhe Ser Gly Asp Lys Asn Glu Leu Ile Ile Gly Ala 620 625 630 gag tcc ttcgtc tcc aat gaa aag atc tac atc gac aag atc gag ttc 2754 Glu Ser Phe ValSer Asn Glu Lys Ile Tyr Ile Asp Lys Ile Glu Phe 635 640 645 atc ccc gtccag ctg tgataggaac tctgattgaa ttctgcatgc gtttggacgt 2809 Ile Pro Val GlnLeu 650 atgctcattc aggttggagc caatttggtt gatgtgtgtg cgagttcttgcgagtctgat 2869 gagacatctc tgtattgtgt ttctttcccc agtgttttct gtacttgtgtaatcggctaa 2929 tcgccaacag attcggcgat gaataaatga gaaataaatt gttctgattttgagtgcaaa 2989 aaaaaaggaa ttagatctgt gtgtgttttt tggatccccg gggcggccgc3039 20 653 PRT Artificial Sequence PRT (1)..(653) Cry3Bb1 variant11231mv1 20 Met Ala Asn Pro Asn Asn Arg Ser Glu His Asp Thr Ile Lys ValThr 1 5 10 15 Pro Asn Ser Glu Leu Gln Thr Asn His Asn Gln Tyr Pro LeuAla Asp 20 25 30 Asn Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr Lys Glu PheLeu Arg 35 40 45 Met Thr Glu Asp Ser Ser Thr Glu Val Leu Asp Asn Ser ThrVal Lys 50 55 60 Asp Ala Val Gly Thr Gly Ile Ser Val Val Gly Gln Ile LeuGly Val 65 70 75 80 Val Gly Val Pro Phe Ala Gly Ala Leu Thr Ser Phe TyrGln Ser Phe 85 90 95 Leu Asn Thr Ile Trp Pro Ser Asp Ala Asp Pro Trp LysAla Phe Met 100 105 110 Ala Gln Val Glu Val Leu Ile Asp Lys Lys Ile GluGlu Tyr Ala Lys 115 120 125 Ser Lys Ala Leu Ala Glu Leu Gln Gly Leu GlnAsn Asn Phe Glu Asp 130 135 140 Tyr Val Asn Ala Leu Asn Ser Trp Lys LysThr Pro Leu Ser Leu Arg 145 150 155 160 Ser Lys Arg Ser Gln Gly Arg IleArg Glu Leu Phe Ser Gln Ala Glu 165 170 175 Ser His Phe Arg Asn Ser MetPro Ser Phe Ala Val Ser Lys Phe Glu 180 185 190 Val Leu Phe Leu Pro ThrTyr Ala Gln Ala Ala Asn Thr His Leu Leu 195 200 205 Leu Leu Lys Asp AlaGln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser 210 215 220 Glu Asp Val AlaGlu Phe Tyr Arg Arg Gln Leu Lys Leu Thr Gln Gln 225 230 235 240 Tyr ThrAsp His Cys Val Asn Trp Tyr Asn Val Gly Leu Asn Gly Leu 245 250 255 ArgGly Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg 260 265 270Glu Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr 275 280285 Asp Ile Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr Arg Asp 290295 300 Ile Phe Thr Asp Pro Ile Phe Leu Leu Thr Thr Leu Gln Lys Tyr Gly305 310 315 320 Pro Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg Lys Pro HisLeu Phe 325 330 335 Asp Tyr Leu Gln Gly Ile Glu Phe His Thr Arg Leu ArgPro Gly Tyr 340 345 350 Phe Gly Lys Asp Ser Phe Asn Tyr Trp Ser Gly AsnTyr Val Glu Thr 355 360 365 Arg Pro Ser Ile Gly Ser Ser Lys Thr Ile ThrSer Pro Phe Tyr Gly 370 375 380 Asp Lys Ser Thr Glu Pro Val Gln Lys LeuSer Phe Asp Gly Gln Lys 385 390 395 400 Val Tyr Arg Thr Ile Ala Asn ThrAsp Val Ala Ala Trp Pro Asn Gly 405 410 415 Lys Val Tyr Leu Gly Val ThrLys Val Asp Phe Ser Gln Tyr Asp Asp 420 425 430 Gln Lys Asn Glu Thr SerThr Gln Thr Tyr Asp Ser Lys Arg Asn Asn 435 440 445 Gly His Val Ser AlaGln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr 450 455 460 Thr Asp Glu ProLeu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala 465 470 475 480 Glu CysPhe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr 485 490 495 TrpThr His Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys 500 505 510Ile Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala 515 520525 Ser Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu Phe Leu 530535 540 Lys Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr Leu Asn Ser545 550 555 560 Ala Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile Arg Tyr AlaSer Thr 565 570 575 Thr Asn Leu Arg Leu Phe Val Gln Asn Ser Asn Asn AspPhe Leu Val 580 585 590 Ile Tyr Ile Asn Lys Thr Met Asn Lys Asp Asp AspLeu Thr Tyr Gln 595 600 605 Thr Phe Asp Leu Ala Thr Thr Asn Ser Asn MetGly Phe Ser Gly Asp 610 615 620 Lys Asn Glu Leu Ile Ile Gly Ala Glu SerPhe Val Ser Asn Glu Lys 625 630 635 640 Ile Tyr Ile Asp Lys Ile Glu PheIle Pro Val Gln Leu 645 650 21 3039 DNA Artificial Sequence Descriptionof Artificial Sequence expression cassette 21 gcggccgcgt taacaagcttctgacgtaag ggatgacgca cctgacgtaa gggatgacgc 60 acctgacgta agggatgacgcacctgacgt aagggatgac gcactcgaga tccccatctc 120 cactgacgta agggatgacgcacaatccca ctatccttcg caagaccctt cctctatata 180 aggaagttca tttcatttggagaggacacg ctgacaagct agcttggctg caggtagatc 240 ctagaaccat cttccacacactcaagccac actattggag aacacacagg gacaacacac 300 cataagatcc aagggaggcctccgccgccg ccggtaacca ccccgcccct ctcctctttc 360 tttctccgtt tttttttccgtctcggtctc gatctttggc cttggtagtt tgggtgggcg 420 agaggcggct tcgtgcgcgcccagatcggt gcgcgggagg ggcgggatct cgcggctggg 480 gctctcgccg gcgtggatccggcccggatc tcgcggggaa tggggctctc ggatgtagat 540 ctgcgatccg ccgttgttgggggagatgat ggggggttta aaatttccgc cgtgctaaac 600 aagatcagga agaggggaaaagggcactat ggtttatatt tttatatatt tctgctgctt 660 cgtcaggctt agatgtgctagatctttctt tcttcttttt gtgggtagaa tttgaatccc 720 tcagcattgt tcatcggtagtttttctttt catgatttgt gacaaatgca gcctcgtgcg 780 gagctttttt gtaggtagaagtgatcaacc atg gcc aac ccc aac aat cgc tcc 834 Met Ala Asn Pro Asn AsnArg Ser 1 5 gag cac gac acg atc aag gtc acc ccc aac tcc gag ctc cag accaac 882 Glu His Asp Thr Ile Lys Val Thr Pro Asn Ser Glu Leu Gln Thr Asn10 15 20 cac aac cag tac ccg ctg gcc gac aac ccc aac tcc acc ctg gaa gag930 His Asn Gln Tyr Pro Leu Ala Asp Asn Pro Asn Ser Thr Leu Glu Glu 2530 35 40 ctg aac tac aag gag ttc ctg cgc atg acc gag gac tcc tcc acg gag978 Leu Asn Tyr Lys Glu Phe Leu Arg Met Thr Glu Asp Ser Ser Thr Glu 4550 55 gtc ctg gac aac tcc acc gtc aag gac gcc gtc ggg acc ggc atc tcc1026 Val Leu Asp Asn Ser Thr Val Lys Asp Ala Val Gly Thr Gly Ile Ser 6065 70 gtc gtt ggg cag atc ctg ggc gtc gtt ggc gtc ccc ttc gca ggt gct1074 Val Val Gly Gln Ile Leu Gly Val Val Gly Val Pro Phe Ala Gly Ala 7580 85 ctc acc tcc ttc tac cag tcc ttc ctg aac acc atc tgg ccc tcc gac1122 Leu Thr Ser Phe Tyr Gln Ser Phe Leu Asn Thr Ile Trp Pro Ser Asp 9095 100 gcc gac ccc tgg aag gcc ttc atg gcc caa gtc gaa gtc ctg atc gac1170 Ala Asp Pro Trp Lys Ala Phe Met Ala Gln Val Glu Val Leu Ile Asp 105110 115 120 aag aag atc gag gag tac gcc aag tcc aag gcc ctg gcc gag ctgcaa 1218 Lys Lys Ile Glu Glu Tyr Ala Lys Ser Lys Ala Leu Ala Glu Leu Gln125 130 135 ggc ctg caa aac aac ttc gag gac tac gtc aac gcg ctg aac tcctgg 1266 Gly Leu Gln Asn Asn Phe Glu Asp Tyr Val Asn Ala Leu Asn Ser Trp140 145 150 aag aag acg cct ctg tcc ctg cgc tcc aag cgc tcc cag gac cgcatc 1314 Lys Lys Thr Pro Leu Ser Leu Arg Ser Lys Arg Ser Gln Asp Arg Ile155 160 165 cgc gag ctg ttc tcc cag gcc gag tcc cac ttc cgc aac tcc atgccg 1362 Arg Glu Leu Phe Ser Gln Ala Glu Ser His Phe Arg Asn Ser Met Pro170 175 180 tcc ttc gcc gtc tcc aag ttc gag gtc ctg ttc ctg ccc acc tacgcc 1410 Ser Phe Ala Val Ser Lys Phe Glu Val Leu Phe Leu Pro Thr Tyr Ala185 190 195 200 cag gct gcc aac acc cac ctc ctg ttg ctg aag gac gcc caggtc ttc 1458 Gln Ala Ala Asn Thr His Leu Leu Leu Leu Lys Asp Ala Gln ValPhe 205 210 215 ggc gag gaa tgg ggc tac tcc tcg gag gac gtc gcc gag ttctac cgt 1506 Gly Glu Glu Trp Gly Tyr Ser Ser Glu Asp Val Ala Glu Phe TyrArg 220 225 230 cgc cag ctg aag ctg acc caa cag tac acc gac cac tgc gtcaac tgg 1554 Arg Gln Leu Lys Leu Thr Gln Gln Tyr Thr Asp His Cys Val AsnTrp 235 240 245 tac aac gtc ggc ctg aac ggc ctg agg ggc tcc acc tac gacgca tgg 1602 Tyr Asn Val Gly Leu Asn Gly Leu Arg Gly Ser Thr Tyr Asp AlaTrp 250 255 260 gtc aag ttc aac cgc ttc cgc agg gag atg acc ctg acc gtcctg gac 1650 Val Lys Phe Asn Arg Phe Arg Arg Glu Met Thr Leu Thr Val LeuAsp 265 270 275 280 ctg atc gtc ctg ttc ccc ttc tac gac atc cgc ctg tactcc aag ggc 1698 Leu Ile Val Leu Phe Pro Phe Tyr Asp Ile Arg Leu Tyr SerLys Gly 285 290 295 gtc aag acc gag ctg acc cgc gac atc ttc acg gac cccatc ttc ctg 1746 Val Lys Thr Glu Leu Thr Arg Asp Ile Phe Thr Asp Pro IlePhe Leu 300 305 310 ctc acg acc ctc cag aag tac ggt ccc acc ttc ctg tccatc gag aac 1794 Leu Thr Thr Leu Gln Lys Tyr Gly Pro Thr Phe Leu Ser IleGlu Asn 315 320 325 tcc atc cgc aag ccc cac ctg ttc gac tac ctc cag ggcatc gag ttc 1842 Ser Ile Arg Lys Pro His Leu Phe Asp Tyr Leu Gln Gly IleGlu Phe 330 335 340 cac acg cgc ctg agg cca ggc tac ttc ggc aag gac tccttc aac tac 1890 His Thr Arg Leu Arg Pro Gly Tyr Phe Gly Lys Asp Ser PheAsn Tyr 345 350 355 360 tgg tcc ggc aac tac gtc gag acc agg ccc tcc atcggc tcc tcg aag 1938 Trp Ser Gly Asn Tyr Val Glu Thr Arg Pro Ser Ile GlySer Ser Lys 365 370 375 acg atc acc tcc cct ttc tac ggc gac aag tcc accgag ccc gtc cag 1986 Thr Ile Thr Ser Pro Phe Tyr Gly Asp Lys Ser Thr GluPro Val Gln 380 385 390 aag ctg tcc ttc gac ggc cag aag gtc tac cgc accatc gcc aac acc 2034 Lys Leu Ser Phe Asp Gly Gln Lys Val Tyr Arg Thr IleAla Asn Thr 395 400 405 gac gtc gcg gct tgg ccg aac ggc aag gtc tac ctgggc gtc acg aag 2082 Asp Val Ala Ala Trp Pro Asn Gly Lys Val Tyr Leu GlyVal Thr Lys 410 415 420 gtc gac ttc tcc cag tac gat gac cag aag aat gaaacc tcc acc cag 2130 Val Asp Phe Ser Gln Tyr Asp Asp Gln Lys Asn Glu ThrSer Thr Gln 425 430 435 440 acc tac gac tcc aag cgc aac aat ggc cac gtctcc gcc cag gac tcc 2178 Thr Tyr Asp Ser Lys Arg Asn Asn Gly His Val SerAla Gln Asp Ser 445 450 455 atc gac cag ctg ccg cct gag acc act gac gagccc ctg gag aag gcc 2226 Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp Glu ProLeu Glu Lys Ala 460 465 470 tac tcc cac cag ctg aac tac gcg gag tgc ttcctg atg caa gac cgc 2274 Tyr Ser His Gln Leu Asn Tyr Ala Glu Cys Phe LeuMet Gln Asp Arg 475 480 485 agg ggc acc atc ccc ttc ttc acc tgg acc caccgc tcc gtc gac ttc 2322 Arg Gly Thr Ile Pro Phe Phe Thr Trp Thr His ArgSer Val Asp Phe 490 495 500 ttc aac acc atc gac gcc gag aag atc acc cagctg ccc gtg gtc aag 2370 Phe Asn Thr Ile Asp Ala Glu Lys Ile Thr Gln LeuPro Val Val Lys 505 510 515 520 gcc tac gcc ctg tcc tcg ggt gcc tcc atcatt gag ggt cca ggc ttc 2418 Ala Tyr Ala Leu Ser Ser Gly Ala Ser Ile IleGlu Gly Pro Gly Phe 525 530 535 acc ggt ggc aac ctg ctg ttc ctg aag gagtcc tcg aac tcc atc gcc 2466 Thr Gly Gly Asn Leu Leu Phe Leu Lys Glu SerSer Asn Ser Ile Ala 540 545 550 aag ttc aag gtc acc ctg aac tcc gct gccttg ctg caa cgc tac cgc 2514 Lys Phe Lys Val Thr Leu Asn Ser Ala Ala LeuLeu Gln Arg Tyr Arg 555 560 565 gtc cgc atc cgc tac gcc tcc acc acg aacctg cgc ctg ttc gtc cag 2562 Val Arg Ile Arg Tyr Ala Ser Thr Thr Asn LeuArg Leu Phe Val Gln 570 575 580 aac tcc aac aat gac ttc ctg gtc atc tacatc aac aag acc atg aac 2610 Asn Ser Asn Asn Asp Phe Leu Val Ile Tyr IleAsn Lys Thr Met Asn 585 590 595 600 aag gac gat gac ctg acc tac cag accttc gac ctc gcc acc acg aac 2658 Lys Asp Asp Asp Leu Thr Tyr Gln Thr PheAsp Leu Ala Thr Thr Asn 605 610 615 tcc aac atg ggc ttc tcg ggc gac aagaat gaa ctg atc att ggt gct 2706 Ser Asn Met Gly Phe Ser Gly Asp Lys AsnGlu Leu Ile Ile Gly Ala 620 625 630 gag tcc ttc gtc tcc aat gaa aag atctac atc gac aag atc gag ttc 2754 Glu Ser Phe Val Ser Asn Glu Lys Ile TyrIle Asp Lys Ile Glu Phe 635 640 645 atc ccc gtc cag ctg tgataggaactctgattgaa ttctgcatgc gtttggacgt 2809 Ile Pro Val Gln Leu 650 atgctcattcaggttggagc caatttggtt gatgtgtgtg cgagttcttg cgagtctgat 2869 gagacatctctgtattgtgt ttctttcccc agtgttttct gtacttgtgt aatcggctaa 2929 tcgccaacagattcggcgat gaataaatga gaaataaatt gttctgattt tgagtgcaaa 2989 aaaaaaggaattagatctgt gtgtgttttt tggatccccg gggcggccgc 3039 22 653 PRT ArtificialSequence PRT (1)..(653) Cry3Bb1 variant 11231mv2 22 Met Ala Asn Pro AsnAsn Arg Ser Glu His Asp Thr Ile Lys Val Thr 1 5 10 15 Pro Asn Ser GluLeu Gln Thr Asn His Asn Gln Tyr Pro Leu Ala Asp 20 25 30 Asn Pro Asn SerThr Leu Glu Glu Leu Asn Tyr Lys Glu Phe Leu Arg 35 40 45 Met Thr Glu AspSer Ser Thr Glu Val Leu Asp Asn Ser Thr Val Lys 50 55 60 Asp Ala Val GlyThr Gly Ile Ser Val Val Gly Gln Ile Leu Gly Val 65 70 75 80 Val Gly ValPro Phe Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser Phe 85 90 95 Leu Asn ThrIle Trp Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe Met 100 105 110 Ala GlnVal Glu Val Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala Lys 115 120 125 SerLys Ala Leu Ala Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu Asp 130 135 140Tyr Val Asn Ala Leu Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu Arg 145 150155 160 Ser Lys Arg Ser Gln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu165 170 175 Ser His Phe Arg Asn Ser Met Pro Ser Phe Ala Val Ser Lys PheGlu 180 185 190 Val Leu Phe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr HisLeu Leu 195 200 205 Leu Leu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp GlyTyr Ser Ser 210 215 220 Glu Asp Val Ala Glu Phe Tyr Arg Arg Gln Leu LysLeu Thr Gln Gln 225 230 235 240 Tyr Thr Asp His Cys Val Asn Trp Tyr AsnVal Gly Leu Asn Gly Leu 245 250 255 Arg Gly Ser Thr Tyr Asp Ala Trp ValLys Phe Asn Arg Phe Arg Arg 260 265 270 Glu Met Thr Leu Thr Val Leu AspLeu Ile Val Leu Phe Pro Phe Tyr 275 280 285 Asp Ile Arg Leu Tyr Ser LysGly Val Lys Thr Glu Leu Thr Arg Asp 290 295 300 Ile Phe Thr Asp Pro IlePhe Leu Leu Thr Thr Leu Gln Lys Tyr Gly 305 310 315 320 Pro Thr Phe LeuSer Ile Glu Asn Ser Ile Arg Lys Pro His Leu Phe 325 330 335 Asp Tyr LeuGln Gly Ile Glu Phe His Thr Arg Leu Arg Pro Gly Tyr 340 345 350 Phe GlyLys Asp Ser Phe Asn Tyr Trp Ser Gly Asn Tyr Val Glu Thr 355 360 365 ArgPro Ser Ile Gly Ser Ser Lys Thr Ile Thr Ser Pro Phe Tyr Gly 370 375 380Asp Lys Ser Thr Glu Pro Val Gln Lys Leu Ser Phe Asp Gly Gln Lys 385 390395 400 Val Tyr Arg Thr Ile Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly405 410 415 Lys Val Tyr Leu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr AspAsp 420 425 430 Gln Lys Asn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys ArgAsn Asn 435 440 445 Gly His Val Ser Ala Gln Asp Ser Ile Asp Gln Leu ProPro Glu Thr 450 455 460 Thr Asp Glu Pro Leu Glu Lys Ala Tyr Ser His GlnLeu Asn Tyr Ala 465 470 475 480 Glu Cys Phe Leu Met Gln Asp Arg Arg GlyThr Ile Pro Phe Phe Thr 485 490 495 Trp Thr His Arg Ser Val Asp Phe PheAsn Thr Ile Asp Ala Glu Lys 500 505 510 Ile Thr Gln Leu Pro Val Val LysAla Tyr Ala Leu Ser Ser Gly Ala 515 520 525 Ser Ile Ile Glu Gly Pro GlyPhe Thr Gly Gly Asn Leu Leu Phe Leu 530 535 540 Lys Glu Ser Ser Asn SerIle Ala Lys Phe Lys Val Thr Leu Asn Ser 545 550 555 560 Ala Ala Leu LeuGln Arg Tyr Arg Val Arg Ile Arg Tyr Ala Ser Thr 565 570 575 Thr Asn LeuArg Leu Phe Val Gln Asn Ser Asn Asn Asp Phe Leu Val 580 585 590 Ile TyrIle Asn Lys Thr Met Asn Lys Asp Asp Asp Leu Thr Tyr Gln 595 600 605 ThrPhe Asp Leu Ala Thr Thr Asn Ser Asn Met Gly Phe Ser Gly Asp 610 615 620Lys Asn Glu Leu Ile Ile Gly Ala Glu Ser Phe Val Ser Asn Glu Lys 625 630635 640 Ile Tyr Ile Asp Lys Ile Glu Phe Ile Pro Val Gln Leu 645 650 233469 DNA Artificial Sequence Description of Artificial Sequenceexpression cassette 23 gcggccgcgt taacaagctt ctgcaggtcc gatgtgagacttttcaacaa agggtaatat 60 ccggaaacct cctcggattc cattgcccag ctatctgtcactttattgtg aagatagtgg 120 aaaaggaagg tggctcctac aaatgccatc attgcgataaaggaaaggcc atcgttgaag 180 atgcctctgc cgacagtggt cccaaagatg gacccccacccacgaggagc atcgtggaaa 240 aagaagacgt tccaaccacg tcttcaaagc aagtggattgatgtgatggt ccgatgtgag 300 acttttcaac aaagggtaat atccggaaac ctcctcggattccattgccc agctatctgt 360 cactttattg tgaagatagt ggaaaaggaa ggtggctcctacaaatgcca tcattgcgat 420 aaaggaaagg ccatcgttga agatgcctct gccgacagtggtcccaaaga tggaccccca 480 cccacgagga gcatcgtgga aaaagaagac gttccaaccacgtcttcaaa gcaagtggat 540 tgatgtgata tctccactga cgtaagggat gacgcacaatcccactatcc ttcgcaagac 600 ccttcctcta tataaggaag ttcatttcat ttggagaggacacgctgaca agctgactct 660 agcagatcct ctagaaccat cttccacaca ctcaagccacactattggag aacacacagg 720 gacaacacac cataagatcc aagggaggcc tccgccgccgccggtaacca ccccgcccct 780 ctcctctttc tttctccgtt tttttttccg tctcggtctcgatctttggc cttggtagtt 840 tgggtgggcg agaggcggct tcgtgcgcgc ccagatcggtgcgcgggagg ggcgggatct 900 cgcggctggg gctctcgccg gcgtggatcc ggcccggatctcgcggggaa tggggctctc 960 ggatgtagat ctgcgatccg ccgttgttgg gggagatgatggggggttta aaatttccgc 1020 cgtgctaaac aagatcagga agaggggaaa agggcactatggtttatatt tttatatatt 1080 tctgctgctt cgtcaggctt agatgtgcta gatctttctttcttcttttt gtgggtagaa 1140 tttgaatccc tcagcattgt tcatcggtag tttttcttttcatgatttgt gacaaatgca 1200 gcctcgtgcg gagctttttt gtaggtagaa gtgatcaaccatg gcc aac ccc aac 1255 Met Ala Asn Pro Asn 1 5 aat cgc tcc gag cac gacacg atc aag gtc acc ccc aac tcc gag ctc 1303 Asn Arg Ser Glu His Asp ThrIle Lys Val Thr Pro Asn Ser Glu Leu 10 15 20 cag acc aac cac aac cag tacccg ctg gcc gac aac ccc aac tcc acc 1351 Gln Thr Asn His Asn Gln Tyr ProLeu Ala Asp Asn Pro Asn Ser Thr 25 30 35 ctg gaa gag ctg aac tac aag gagttc ctg cgc atg acc gag gac tcc 1399 Leu Glu Glu Leu Asn Tyr Lys Glu PheLeu Arg Met Thr Glu Asp Ser 40 45 50 tcc acg gag gtc ctg gac aac tcc accgtc aag gac gcc gtc ggg acc 1447 Ser Thr Glu Val Leu Asp Asn Ser Thr ValLys Asp Ala Val Gly Thr 55 60 65 ggc atc tcc gtc gtt ggg cag atc ctg ggcgtc gtt ggc gtc ccc ttc 1495 Gly Ile Ser Val Val Gly Gln Ile Leu Gly ValVal Gly Val Pro Phe 70 75 80 85 gca ggt gct ctc acc tcc ttc tac cag tccttc ctg aac acc atc tgg 1543 Ala Gly Ala Leu Thr Ser Phe Tyr Gln Ser PheLeu Asn Thr Ile Trp 90 95 100 ccc tcc gac gcc gac ccc tgg aag gcc ttcatg gcc caa gtc gaa gtc 1591 Pro Ser Asp Ala Asp Pro Trp Lys Ala Phe MetAla Gln Val Glu Val 105 110 115 ctg atc gac aag aag atc gag gag tac gccaag tcc aag gcc ctg gcc 1639 Leu Ile Asp Lys Lys Ile Glu Glu Tyr Ala LysSer Lys Ala Leu Ala 120 125 130 gag ctg caa ggc ctg caa aac aac ttc gaggac tac gtc aac gcg ctg 1687 Glu Leu Gln Gly Leu Gln Asn Asn Phe Glu AspTyr Val Asn Ala Leu 135 140 145 aac tcc tgg aag aag acg cct ctg tcc ctgcgc tcc aag cgc tcc cag 1735 Asn Ser Trp Lys Lys Thr Pro Leu Ser Leu ArgSer Lys Arg Ser Gln 150 155 160 165 gac cgc atc cgc gag ctg ttc tcc caggcc gag tcc cac ttc cgc aac 1783 Asp Arg Ile Arg Glu Leu Phe Ser Gln AlaGlu Ser His Phe Arg Asn 170 175 180 tcc atg ccg tcc ttc gcc gtc tcc aagttc gag gtc ctg ttc ctg ccc 1831 Ser Met Pro Ser Phe Ala Val Ser Lys PheGlu Val Leu Phe Leu Pro 185 190 195 acc tac gcc cag gct gcc aac acc cacctc ctg ttg ctg aag gac gcc 1879 Thr Tyr Ala Gln Ala Ala Asn Thr His LeuLeu Leu Leu Lys Asp Ala 200 205 210 cag gtc ttc ggc gag gaa tgg ggc tactcc tcg gag gac gtc gcc gag 1927 Gln Val Phe Gly Glu Glu Trp Gly Tyr SerSer Glu Asp Val Ala Glu 215 220 225 ttc tac cgt cgc cag ctg aag ctg acccaa cag tac acc gac cac tgc 1975 Phe Tyr Arg Arg Gln Leu Lys Leu Thr GlnGln Tyr Thr Asp His Cys 230 235 240 245 gtc aac tgg tac aac gtc ggc ctgaac ggc ctg agg ggc tcc acc tac 2023 Val Asn Trp Tyr Asn Val Gly Leu AsnGly Leu Arg Gly Ser Thr Tyr 250 255 260 gac gca tgg gtc aag ttc aac cgcttc cgc agg gag atg acc ctg acc 2071 Asp Ala Trp Val Lys Phe Asn Arg PheArg Arg Glu Met Thr Leu Thr 265 270 275 gtc ctg gac ctg atc gtc ctg ttcccc ttc tac gac atc cgc ctg tac 2119 Val Leu Asp Leu Ile Val Leu Phe ProPhe Tyr Asp Ile Arg Leu Tyr 280 285 290 tcc aag ggc gtc aag acc gag ctgacc cgc gac atc ttc acg gac ccc 2167 Ser Lys Gly Val Lys Thr Glu Leu ThrArg Asp Ile Phe Thr Asp Pro 295 300 305 atc ttc ctg ctc acg acc ctc cagaag tac ggt ccc acc ttc ctg tcc 2215 Ile Phe Leu Leu Thr Thr Leu Gln LysTyr Gly Pro Thr Phe Leu Ser 310 315 320 325 atc gag aac tcc atc cgc aagccc cac ctg ttc gac tac ctc cag ggc 2263 Ile Glu Asn Ser Ile Arg Lys ProHis Leu Phe Asp Tyr Leu Gln Gly 330 335 340 atc gag ttc cac acg cgc ctgagg cca ggc tac ttc ggc aag gac tcc 2311 Ile Glu Phe His Thr Arg Leu ArgPro Gly Tyr Phe Gly Lys Asp Ser 345 350 355 ttc aac tac tgg tcc ggc aactac gtc gag acc agg ccc tcc atc ggc 2359 Phe Asn Tyr Trp Ser Gly Asn TyrVal Glu Thr Arg Pro Ser Ile Gly 360 365 370 tcc tcg aag acg atc acc tcccct ttc tac ggc gac aag tcc acc gag 2407 Ser Ser Lys Thr Ile Thr Ser ProPhe Tyr Gly Asp Lys Ser Thr Glu 375 380 385 ccc gtc cag aag ctg tcc ttcgac ggc cag aag gtc tac cgc acc atc 2455 Pro Val Gln Lys Leu Ser Phe AspGly Gln Lys Val Tyr Arg Thr Ile 390 395 400 405 gcc aac acc gac gtc gcggct tgg ccg aac ggc aag gtc tac ctg ggc 2503 Ala Asn Thr Asp Val Ala AlaTrp Pro Asn Gly Lys Val Tyr Leu Gly 410 415 420 gtc acg aag gtc gac ttctcc cag tac gat gac cag aag aat gaa acc 2551 Val Thr Lys Val Asp Phe SerGln Tyr Asp Asp Gln Lys Asn Glu Thr 425 430 435 tcc acc cag acc tac gactcc aag cgc aac aat ggc cac gtc tcc gcc 2599 Ser Thr Gln Thr Tyr Asp SerLys Arg Asn Asn Gly His Val Ser Ala 440 445 450 cag gac tcc atc gac cagctg ccg cct gag acc act gac gag ccc ctg 2647 Gln Asp Ser Ile Asp Gln LeuPro Pro Glu Thr Thr Asp Glu Pro Leu 455 460 465 gag aag gcc tac tcc caccag ctg aac tac gcg gag tgc ttc ctg atg 2695 Glu Lys Ala Tyr Ser His GlnLeu Asn Tyr Ala Glu Cys Phe Leu Met 470 475 480 485 caa gac cgc agg ggcacc atc ccc ttc ttc acc tgg acc cac cgc tcc 2743 Gln Asp Arg Arg Gly ThrIle Pro Phe Phe Thr Trp Thr His Arg Ser 490 495 500 gtc gac ttc ttc aacacc atc gac gcc gag aag atc acc cag ctg ccc 2791 Val Asp Phe Phe Asn ThrIle Asp Ala Glu Lys Ile Thr Gln Leu Pro 505 510 515 gtg gtc aag gcc tacgcc ctg tcc tcg ggt gcc tcc atc att gag ggt 2839 Val Val Lys Ala Tyr AlaLeu Ser Ser Gly Ala Ser Ile Ile Glu Gly 520 525 530 cca ggc ttc acc ggtggc aac ctg ctg ttc ctg aag gag tcc tcg aac 2887 Pro Gly Phe Thr Gly GlyAsn Leu Leu Phe Leu Lys Glu Ser Ser Asn 535 540 545 tcc atc gcc aag ttcaag gtc acc ctg aac tcc gct gcc ttg ctg caa 2935 Ser Ile Ala Lys Phe LysVal Thr Leu Asn Ser Ala Ala Leu Leu Gln 550 555 560 565 cgc tac cgc gtccgc atc cgc tac gcc tcc acc acg aac ctg cgc ctg 2983 Arg Tyr Arg Val ArgIle Arg Tyr Ala Ser Thr Thr Asn Leu Arg Leu 570 575 580 ttc gtc cag aactcc aac aat gac ttc ctg gtc atc tac atc aac aag 3031 Phe Val Gln Asn SerAsn Asn Asp Phe Leu Val Ile Tyr Ile Asn Lys 585 590 595 acc atg aac aaggac gat gac ctg acc tac cag acc ttc gac ctc gcc 3079 Thr Met Asn Lys AspAsp Asp Leu Thr Tyr Gln Thr Phe Asp Leu Ala 600 605 610 acc acg aac tccaac atg ggc ttc tcg ggc gac aag aat gaa ctg atc 3127 Thr Thr Asn Ser AsnMet Gly Phe Ser Gly Asp Lys Asn Glu Leu Ile 615 620 625 att ggt gct gagtcc ttc gtc tcc aat gaa aag atc tac atc gac aag 3175 Ile Gly Ala Glu SerPhe Val Ser Asn Glu Lys Ile Tyr Ile Asp Lys 630 635 640 645 atc gag ttcatc ccc gtc cag ctg tgataggaac tctgattgaa ttctgcatgc 3229 Ile Glu PheIle Pro Val Gln Leu 650 gtttggacgt atgctcattc aggttggagc caatttggttgatgtgtgtg cgagttcttg 3289 cgagtctgat gagacatctc tgtattgtgt ttctttccccagtgttttct gtacttgtgt 3349 aatcggctaa tcgccaacag attcggcgat gaataaatgagaaataaatt gttctgattt 3409 tgagtgcaaa aaaaaaggaa ttagatctgt gtgtgttttttggatccccg gggcggccgc 3469 24 653 PRT Artificial Sequence PRT (1)..(653)Cry3Bb1 variant 11231mv2 24 Met Ala Asn Pro Asn Asn Arg Ser Glu His AspThr Ile Lys Val Thr 1 5 10 15 Pro Asn Ser Glu Leu Gln Thr Asn His AsnGln Tyr Pro Leu Ala Asp 20 25 30 Asn Pro Asn Ser Thr Leu Glu Glu Leu AsnTyr Lys Glu Phe Leu Arg 35 40 45 Met Thr Glu Asp Ser Ser Thr Glu Val LeuAsp Asn Ser Thr Val Lys 50 55 60 Asp Ala Val Gly Thr Gly Ile Ser Val ValGly Gln Ile Leu Gly Val 65 70 75 80 Val Gly Val Pro Phe Ala Gly Ala LeuThr Ser Phe Tyr Gln Ser Phe 85 90 95 Leu Asn Thr Ile Trp Pro Ser Asp AlaAsp Pro Trp Lys Ala Phe Met 100 105 110 Ala Gln Val Glu Val Leu Ile AspLys Lys Ile Glu Glu Tyr Ala Lys 115 120 125 Ser Lys Ala Leu Ala Glu LeuGln Gly Leu Gln Asn Asn Phe Glu Asp 130 135 140 Tyr Val Asn Ala Leu AsnSer Trp Lys Lys Thr Pro Leu Ser Leu Arg 145 150 155 160 Ser Lys Arg SerGln Asp Arg Ile Arg Glu Leu Phe Ser Gln Ala Glu 165 170 175 Ser His PheArg Asn Ser Met Pro Ser Phe Ala Val Ser Lys Phe Glu 180 185 190 Val LeuPhe Leu Pro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu 195 200 205 LeuLeu Lys Asp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser 210 215 220Glu Asp Val Ala Glu Phe Tyr Arg Arg Gln Leu Lys Leu Thr Gln Gln 225 230235 240 Tyr Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu Asn Gly Leu245 250 255 Arg Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe ArgArg 260 265 270 Glu Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe ProPhe Tyr 275 280 285 Asp Ile Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu LeuThr Arg Asp 290 295 300 Ile Phe Thr Asp Pro Ile Phe Leu Leu Thr Thr LeuGln Lys Tyr Gly 305 310 315 320 Pro Thr Phe Leu Ser Ile Glu Asn Ser IleArg Lys Pro His Leu Phe 325 330 335 Asp Tyr Leu Gln Gly Ile Glu Phe HisThr Arg Leu Arg Pro Gly Tyr 340 345 350 Phe Gly Lys Asp Ser Phe Asn TyrTrp Ser Gly Asn Tyr Val Glu Thr 355 360 365 Arg Pro Ser Ile Gly Ser SerLys Thr Ile Thr Ser Pro Phe Tyr Gly 370 375 380 Asp Lys Ser Thr Glu ProVal Gln Lys Leu Ser Phe Asp Gly Gln Lys 385 390 395 400 Val Tyr Arg ThrIle Ala Asn Thr Asp Val Ala Ala Trp Pro Asn Gly 405 410 415 Lys Val TyrLeu Gly Val Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp 420 425 430 Gln LysAsn Glu Thr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn 435 440 445 GlyHis Val Ser Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr 450 455 460Thr Asp Glu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala 465 470475 480 Glu Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr485 490 495 Trp Thr His Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala GluLys 500 505 510 Ile Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser SerGly Ala 515 520 525 Ser Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn LeuLeu Phe Leu 530 535 540 Lys Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys ValThr Leu Asn Ser 545 550 555 560 Ala Ala Leu Leu Gln Arg Tyr Arg Val ArgIle Arg Tyr Ala Ser Thr 565 570 575 Thr Asn Leu Arg Leu Phe Val Gln AsnSer Asn Asn Asp Phe Leu Val 580 585 590 Ile Tyr Ile Asn Lys Thr Met AsnLys Asp Asp Asp Leu Thr Tyr Gln 595 600 605 Thr Phe Asp Leu Ala Thr ThrAsn Ser Asn Met Gly Phe Ser Gly Asp 610 615 620 Lys Asn Glu Leu Ile IleGly Ala Glu Ser Phe Val Ser Asn Glu Lys 625 630 635 640 Ile Tyr Ile AspLys Ile Glu Phe Ile Pro Val Gln Leu 645 650 25 416 DNA ArtificialSequence Description of Artificial Sequence non-naturally occurringnucleotide sequence encoding Zea mays ribulose bis-phosphate carboxylasechloroplast targeting peptide 25 ttctagagga tcagc atg gcg ccc acc gtgatg atg gcc tcg tcg gcc acc 51 Met Ala Pro Thr Val Met Met Ala Ser SerAla Thr 1 5 10 gcc gtc gct ccg ttc ctg ggg ctc aag tcc acc gcc agc ctcccc gtc 99 Ala Val Ala Pro Phe Leu Gly Leu Lys Ser Thr Ala Ser Leu ProVal 15 20 25 gcc cgc cgc tcc tcc aga agc ctc ggc aac gtc agc aac ggc ggaagg 147 Ala Arg Arg Ser Ser Arg Ser Leu Gly Asn Val Ser Asn Gly Gly Arg30 35 40 atc cgg tgc atg cag gtaacaaatg catcctagct agtagttctt tgcattgcag202 Ile Arg Cys Met Gln 45 cagctgcagc tagcgagtta gtaataggaa gggaactgatgatccatgca tggactgatg 262 tgtgttgccc atcccatccc atcccatttc ccaaacgaaccgaaaacacc gtactacgtg 322 cag gtg tgg ccc tac ggc aac aag aag ttc gagacg ctg tcg tac ctg 370 Val Trp Pro Tyr Gly Asn Lys Lys Phe Glu Thr LeuSer Tyr Leu 50 55 60 ccg ccg ctg tcg acc ggc ggg cgc atc cgc tgc atg caggcc atg g 416 Pro Pro Leu Ser Thr Gly Gly Arg Ile Arg Cys Met Gln AlaMet 65 70 75 26 79 PRT Artificial Sequence PRT (1)..(48) full length zeamays transit peptide 26 Met Ala Pro Thr Val Met Met Ala Ser Ser Ala ThrAla Val Ala Pro 1 5 10 15 Phe Leu Gly Leu Lys Ser Thr Ala Ser Leu ProVal Ala Arg Arg Ser 20 25 30 Ser Arg Ser Leu Gly Asn Val Ser Asn Gly GlyArg Ile Arg Cys Met 35 40 45 Gln Val Trp Pro Tyr Gly Asn Lys Lys Phe GluThr Leu Ser Tyr Leu 50 55 60 Pro Pro Leu Ser Thr Gly Gly Arg Ile Arg CysMet Gln Ala Met 65 70 75 27 49 PRT Artificial Sequence PRT (1)..(49) Zeamays targeting peptide sequence encoded 5′ of the intronic sequenceindicated in SEQID NO25 27 Met Ala Pro Thr Val Met Met Ala Ser Ser AlaThr Ala Val Ala Pro 1 5 10 15 Phe Leu Gly Leu Lys Ser Thr Ala Ser LeuPro Val Ala Arg Arg Ser 20 25 30 Ser Arg Ser Leu Gly Asn Val Ser Asn GlyGly Arg Ile Arg Cys Met 35 40 45 Gln 28 30 PRT Artificial Sequence PRT(1)..(30) Zea mays targeting peptide sequence encoded 3′ of the intronicsequence indicated in SEQID NO25 28 Val Trp Pro Tyr Gly Asn Lys Lys PheGlu Thr Leu Ser Tyr Leu Pro 1 5 10 15 Pro Leu Ser Thr Gly Gly Arg IleArg Cys Met Gln Ala Met 20 25 30 29 202 DNA Cauliflower mosaic virus PRT(1)..(30) a cauliflower mosaic virus 35S promoter sequence, P-CaMV.35S29 gacgcacctg acgtaaggga tgacgcacct gacgtaaggg atgacgcacc tgacgtaagg 60gatgacgcac tcgagatccc catctccact gacgtaaggg atgacgcaca atcccactat 120ccttcgcaag acccttcctc tatataagga agttcatttc atttggagag gacacgctga 180caagctagct tggctgcagg ta 202 30 416 DNA Artificial Sequence Descriptionof Artificial Sequence modified cauliflower mosaic virus promoter AS4 30ttctagagga tcagcatggc gcccaccgtg atgatggcct cgtcggccac cgccgtcgct 60ccgttcctgg ggctcaagtc caccgccagc ctccccgtcg cccgccgctc ctccagaagc 120ctcggcaacg tcagcaacgg cggaaggatc cggtgcatgc aggtaacaaa tgcatcctag 180ctagtagttc tttgcattgc agcagctgca gctagcgagt tagtaatagg aagggaactg 240atgatccatg catggactga tgtgtgttgc ccatcccatc ccatcccatt tcccaaacga 300accgaaaaca ccgtactacg tgcaggtgtg gccctacggc aacaagaagt tcgagacgct 360gtcgtacctg ccgccgctgt cgaccggcgg gcgcatccgc tgcatgcagg ccatgg 416 31 75DNA Triticum aestivum 31 ctagaaccat cttccacaca ctcaagccac actattggagaacacacagg gacaacacac 60 cataagatcc aaggg 75 32 804 DNA Oryza sp. 32accgtcttcg gtacgcgctc actccgccct ctgcctttgt tactgccacg tttctctgaa 60tgctctcttg tgtggtgatt gctgagagtg gtttagctgg atctagaatt acactctgaa 120atcgtgttct gcctgtgctg attacttgcc gtcctttgta gcagcaaaat atagggacat 180ggtagtacga aacgaagata gaacctacac agcaatacga gaaatgtgta atttggtgct 240tagcggtatt tatttaagca catgttggtg ttatagggca cttggattca gaagtttgct 300gttaatttag gcacaggctt catactacat gggtcaatag tatagggatt catattatag 360gcgatactat aataatttgt tcgtctgcag agcttattat ttgccaaaat tagatattcc 420tattctgttt ttgtttgtgt gctgttaaat tgttaacgcc tgaaggaata aatataaatg 480acgaaatttt gatgtttatc tctgctcctt tattgtgacc ataagtcaag atcagatgca 540cttgttttaa atattgttgt ctgaagaaat aagtactgac agtattttga tgcattgatc 600tgcttgtttg ttgtaacaaa atttaaaaat aaagagtttc ctttttgttg ctctccttac 660ctcctgatgg tatctagtat ctaccaactg acactatatt gcttctcttt acatacgtat 720cttgctcgat gccttctccc tagtgttgac cagtgttact cacatagtct ttgctcattt 780cattgtaatg cagataccaa gcgg 804 33 804 DNA Zea mays 33 accgtcttcggtacgcgctc actccgccct ctgcctttgt tactgccacg tttctctgaa 60 tgctctcttgtgtggtgatt gctgagagtg gtttagctgg atctagaatt acactctgaa 120 atcgtgttctgcctgtgctg attacttgcc gtcctttgta gcagcaaaat atagggacat 180 ggtagtacgaaacgaagata gaacctacac agcaatacga gaaatgtgta atttggtgct 240 tagcggtatttatttaagca catgttggtg ttatagggca cttggattca gaagtttgct 300 gttaatttaggcacaggctt catactacat gggtcaatag tatagggatt catattatag 360 gcgatactataataatttgt tcgtctgcag agcttattat ttgccaaaat tagatattcc 420 tattctgtttttgtttgtgt gctgttaaat tgttaacgcc tgaaggaata aatataaatg 480 acgaaattttgatgtttatc tctgctcctt tattgtgacc ataagtcaag atcagatgca 540 cttgttttaaatattgttgt ctgaagaaat aagtactgac agtattttga tgcattgatc 600 tgcttgtttgttgtaacaaa atttaaaaat aaagagtttc ctttttgttg ctctccttac 660 ctcctgatggtatctagtat ctaccaactg acactatatt gcttctcttt acatacgtat 720 cttgctcgatgccttctccc tagtgttgac cagtgttact cacatagtct ttgctcattt 780 cattgtaatgcagataccaa gcgg 804 34 257 DNA Agrobacterium tumefaciens 34 tcccgatcgttcaaacattt ggcaataaag tttcttaaga ttgaatcctg ttgccggtct 60 tgcgatgattatcatataat ttctgttgaa ttacgttaag catgtaataa ttaacatgta 120 atgcatgacgttatttatga gatgggtttt tatgattaga gtcccgcaat tatacattta 180 atacgcgatagaaaacaaaa tatagcgcgc aaactaggat aaattatcgc gcgcggtgtc 240 atctatgttactagatc 257 35 234 DNA Triticum aestivum 35 aattctgcat gcgtttggacgtatgctcat tcaggttgga gccaatttgg ttgatgtgtg 60 tgcgagttct tgcgagtctgatgagacatc tctgtattgt gtttctttcc ccagtgtttt 120 ctgtacttgt gtaatcggctaatcgccaac agattcggcg atgaataaat gagaaataaa 180 ttgttctgat tttgagtgcaaaaaaaaagg aattagatct gtgtgtgttt tttg 234 36 3455 DNA ArtificialSequence Description of Artificial Sequence expression cassette 36gcggccgcgt taacaagctt ctgacgtaag ggatgacgca cctgacgtaa gggatgacgc 60acctgacgta agggatgacg cacctgacgt aagggatgac gcactcgaga tccccatctc 120cactgacgta agggatgacg cacaatccca ctatccttcg caagaccctt cctctatata 180aggaagttca tttcatttgg agaggacacg ctgacaagct agcttggctg caggtagatc 240ctagaaccat cttccacaca ctcaagccac actattggag aacacacagg gacaacacac 300cataagatcc aagggaggcc tccgccgccg ccggtaacca ccccgcccct ctcctctttc 360tttctccgtt tttttttccg tctcggtctc gatctttggc cttggtagtt tgggtgggcg 420agaggcggct tcgtgcgcgc ccagatcggt gcgcgggagg ggcgggatct cgcggctggg 480gctctcgccg gcgtggatcc ggcccggatc tcgcggggaa tggggctctc ggatgtagat 540ctgcgatccg ccgttgttgg gggagatgat ggggggttta aaatttccgc cgtgctaaac 600aagatcagga agaggggaaa agggcactat ggtttatatt tttatatatt tctgctgctt 660cgtcaggctt agatgtgcta gatctttctt tcttcttttt gtgggtagaa tttgaatccc 720tcagcattgt tcatcggtag tttttctttt catgatttgt gacaaatgca gcctcgtgcg 780gagctttttt gtaggtagaa gtgatcaacc tctagaggat cagcatggcg cccaccgtga 840tgatggcctc gtcggccacc gccgtcgctc cgttcctggg gctcaagtcc accgccagcc 900tccccgtcgc ccgccgctcc tccagaagcc tcggcaacgt cagcaacggc ggaaggatcc 960ggtgcatgca ggtaacaaat gcatcctagc tagtagttct ttgcattgca gcagctgcag 1020ctagcgagtt agtaatagga agggaactga tgatccatgc atggactgat gtgtgttgcc 1080catcccatcc catcccattt cccaaacgaa ccgaaaacac cgtactacgt gcaggtgtgg 1140ccctacggca acaagaagtt cgagacgctg tcgtacctgc cgccgctgtc gaccggcggg 1200cgcatccgct gcatgcaggc c atg gca aac cct aac aat cgt tcc gaa cac 1251 MetAla Asn Pro Asn Asn Arg Ser Glu His 1 5 10 gac acc atc aag gtt act ccaaac tct gag ttg caa act aat cac aac 1299 Asp Thr Ile Lys Val Thr Pro AsnSer Glu Leu Gln Thr Asn His Asn 15 20 25 cag tac cca ttg gct gac aat cctaac agt act ctt gag gaa ctt aac 1347 Gln Tyr Pro Leu Ala Asp Asn Pro AsnSer Thr Leu Glu Glu Leu Asn 30 35 40 tac aag gag ttt ctc cgg atg acc gaagat agc tcc act gag gtt ctc 1395 Tyr Lys Glu Phe Leu Arg Met Thr Glu AspSer Ser Thr Glu Val Leu 45 50 55 gat aac tct aca gtg aag gac gct gtt ggaact ggc att agc gtt gtg 1443 Asp Asn Ser Thr Val Lys Asp Ala Val Gly ThrGly Ile Ser Val Val 60 65 70 gga cag att ctt gga gtg gtt ggt gtt cca ttcgct gga gct ttg acc 1491 Gly Gln Ile Leu Gly Val Val Gly Val Pro Phe AlaGly Ala Leu Thr 75 80 85 90 agc ttc tac cag tcc ttt ctc aac acc atc tggcct tca gat gct gat 1539 Ser Phe Tyr Gln Ser Phe Leu Asn Thr Ile Trp ProSer Asp Ala Asp 95 100 105 ccc tgg aag gct ttc atg gcc caa gtg gaa gtcttg atc gat aag aag 1587 Pro Trp Lys Ala Phe Met Ala Gln Val Glu Val LeuIle Asp Lys Lys 110 115 120 atc gaa gag tat gcc aag tct aaa gcc ttg gctgag ttg caa ggt ttg 1635 Ile Glu Glu Tyr Ala Lys Ser Lys Ala Leu Ala GluLeu Gln Gly Leu 125 130 135 cag aac aac ttc gag gat tac gtc aac gca ctcaac agc tgg aag aaa 1683 Gln Asn Asn Phe Glu Asp Tyr Val Asn Ala Leu AsnSer Trp Lys Lys 140 145 150 act ccc ttg agt ctc agg tct aag cgt tcc caggac cgt att cgt gaa 1731 Thr Pro Leu Ser Leu Arg Ser Lys Arg Ser Gln AspArg Ile Arg Glu 155 160 165 170 ctt ttc agc caa gcc gaa tcc cac ttc agaaac tcc atg cct agc ttt 1779 Leu Phe Ser Gln Ala Glu Ser His Phe Arg AsnSer Met Pro Ser Phe 175 180 185 gcc gtt tct aag ttc gag gtg ctc ttc ttgcca aca tac gca caa gct 1827 Ala Val Ser Lys Phe Glu Val Leu Phe Leu ProThr Tyr Ala Gln Ala 190 195 200 gcc aac act cat ctc ttg ctt ctc aaa gacgct cag gtg ttt ggt gag 1875 Ala Asn Thr His Leu Leu Leu Leu Lys Asp AlaGln Val Phe Gly Glu 205 210 215 gaa tgg ggt tac tcc agt gaa gat gtt gccgag ttc tac cgt agg cag 1923 Glu Trp Gly Tyr Ser Ser Glu Asp Val Ala GluPhe Tyr Arg Arg Gln 220 225 230 ctc aag ttg act caa cag tac aca gac cactgc gtc aac tgg tac aac 1971 Leu Lys Leu Thr Gln Gln Tyr Thr Asp His CysVal Asn Trp Tyr Asn 235 240 245 250 gtt ggg ctc aat ggt ctt aga gga tctacc tac gac gca tgg gtg aag 2019 Val Gly Leu Asn Gly Leu Arg Gly Ser ThrTyr Asp Ala Trp Val Lys 255 260 265 ttc aac agg ttt cgt aga gag atg accttg act gtg ctc gat ctt atc 2067 Phe Asn Arg Phe Arg Arg Glu Met Thr LeuThr Val Leu Asp Leu Ile 270 275 280 gtt ctc ttt cca ttc tac gac att cgtctt tac tcc aaa ggc gtt aag 2115 Val Leu Phe Pro Phe Tyr Asp Ile Arg LeuTyr Ser Lys Gly Val Lys 285 290 295 aca gag ctg acc aga gac atc ttc accgat ccc atc ttc cta ctt acg 2163 Thr Glu Leu Thr Arg Asp Ile Phe Thr AspPro Ile Phe Leu Leu Thr 300 305 310 acc ctg cag aaa tac ggt cca act tttctc tcc att gag aac agc atc 2211 Thr Leu Gln Lys Tyr Gly Pro Thr Phe LeuSer Ile Glu Asn Ser Ile 315 320 325 330 agg aag cct cac ctc ttc gac tatctg caa ggc att gag ttt cac acc 2259 Arg Lys Pro His Leu Phe Asp Tyr LeuGln Gly Ile Glu Phe His Thr 335 340 345 agg ttg caa cct ggt tac ttc ggtaag gat tcc ttc aac tac tgg agc 2307 Arg Leu Gln Pro Gly Tyr Phe Gly LysAsp Ser Phe Asn Tyr Trp Ser 350 355 360 gga aac tac gtt gaa acc aga ccatcc atc gga tct agc aag acc atc 2355 Gly Asn Tyr Val Glu Thr Arg Pro SerIle Gly Ser Ser Lys Thr Ile 365 370 375 act tct cca ttc tac ggt gac aagagc act gag cca gtg cag aag ttg 2403 Thr Ser Pro Phe Tyr Gly Asp Lys SerThr Glu Pro Val Gln Lys Leu 380 385 390 agc ttc gat ggg cag aag gtg tataga acc atc gcc aat acc gat gtt 2451 Ser Phe Asp Gly Gln Lys Val Tyr ArgThr Ile Ala Asn Thr Asp Val 395 400 405 410 gca gct tgg cct aat ggc aaggtc tac ctt gga gtt act aaa gtg gac 2499 Ala Ala Trp Pro Asn Gly Lys ValTyr Leu Gly Val Thr Lys Val Asp 415 420 425 ttc tcc caa tac gac gat cagaag aac gag aca tct act caa acc tac 2547 Phe Ser Gln Tyr Asp Asp Gln LysAsn Glu Thr Ser Thr Gln Thr Tyr 430 435 440 gat agt aag agg aac aat ggccat gtt tcc gca caa gac tcc att gac 2595 Asp Ser Lys Arg Asn Asn Gly HisVal Ser Ala Gln Asp Ser Ile Asp 445 450 455 caa ctt cca cct gaa acc actgat gaa cca ttg gag aag gct tac agt 2643 Gln Leu Pro Pro Glu Thr Thr AspGlu Pro Leu Glu Lys Ala Tyr Ser 460 465 470 cac caa ctt aac tac gcc gaatgc ttt ctc atg caa gac agg cgt ggc 2691 His Gln Leu Asn Tyr Ala Glu CysPhe Leu Met Gln Asp Arg Arg Gly 475 480 485 490 acc att ccg ttc ttt acatgg act cac agg tct gtc gac ttc ttt aac 2739 Thr Ile Pro Phe Phe Thr TrpThr His Arg Ser Val Asp Phe Phe Asn 495 500 505 act atc gac gct gag aagatt acc caa ctt ccc gtg gtc aag gct tat 2787 Thr Ile Asp Ala Glu Lys IleThr Gln Leu Pro Val Val Lys Ala Tyr 510 515 520 gcc ttg tcc agc gga gcttcc atc att gaa ggt cca ggc ttc acc ggt 2835 Ala Leu Ser Ser Gly Ala SerIle Ile Glu Gly Pro Gly Phe Thr Gly 525 530 535 ggc aac ttg ctc ttc cttaag gag tcc agc aac tcc atc gcc aag ttc 2883 Gly Asn Leu Leu Phe Leu LysGlu Ser Ser Asn Ser Ile Ala Lys Phe 540 545 550 aaa gtg aca ctt aac tcagca gcc ttg ctc caa cgt tac agg gtt cgt 2931 Lys Val Thr Leu Asn Ser AlaAla Leu Leu Gln Arg Tyr Arg Val Arg 555 560 565 570 atc aga tac gca agcact acc aat ctt cgc ctc ttt gtc cag aac agc 2979 Ile Arg Tyr Ala Ser ThrThr Asn Leu Arg Leu Phe Val Gln Asn Ser 575 580 585 aac aat gat ttc cttgtc atc tac atc aac aag act atg aac aaa gac 3027 Asn Asn Asp Phe Leu ValIle Tyr Ile Asn Lys Thr Met Asn Lys Asp 590 595 600 gat gac ctc acc taccaa aca ttc gat ctt gcc act acc aat agt aac 3075 Asp Asp Leu Thr Tyr GlnThr Phe Asp Leu Ala Thr Thr Asn Ser Asn 605 610 615 atg gga ttc tct ggtgac aag aac gag ctg atc ata ggt gct gag agc 3123 Met Gly Phe Ser Gly AspLys Asn Glu Leu Ile Ile Gly Ala Glu Ser 620 625 630 ttt gtc tct aat gagaag att tac ata gac aag atc gag ttc att cca 3171 Phe Val Ser Asn Glu LysIle Tyr Ile Asp Lys Ile Glu Phe Ile Pro 635 640 645 650 gtt caa ctctaatagatcc cccgggctgc aggaattctg catgcgtttg 3220 Val Gln Leu gacgtatgctcattcaggtt ggagccaatt tggttgatgt gtgtgcgagt tcttgcgagt 3280 ctgatgagacatctctgtat tgtgtttctt tccccagtgt tttctgtact tgtgtaatcg 3340 gctaatcgccaacagattcg gcgatgaata aatgagaaat aaattgttct gattttgagt 3400 gcaaaaaaaaaggaattaga tctgtgtgtg ttttttggat ccccggggcg gccgc 3455 37 653 PRTArtificial Sequence PRT (1)..(653) variant Cry3BB1 coding sequenceencoding v11231 37 Met Ala Asn Pro Asn Asn Arg Ser Glu His Asp Thr IleLys Val Thr 1 5 10 15 Pro Asn Ser Glu Leu Gln Thr Asn His Asn Gln TyrPro Leu Ala Asp 20 25 30 Asn Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr LysGlu Phe Leu Arg 35 40 45 Met Thr Glu Asp Ser Ser Thr Glu Val Leu Asp AsnSer Thr Val Lys 50 55 60 Asp Ala Val Gly Thr Gly Ile Ser Val Val Gly GlnIle Leu Gly Val 65 70 75 80 Val Gly Val Pro Phe Ala Gly Ala Leu Thr SerPhe Tyr Gln Ser Phe 85 90 95 Leu Asn Thr Ile Trp Pro Ser Asp Ala Asp ProTrp Lys Ala Phe Met 100 105 110 Ala Gln Val Glu Val Leu Ile Asp Lys LysIle Glu Glu Tyr Ala Lys 115 120 125 Ser Lys Ala Leu Ala Glu Leu Gln GlyLeu Gln Asn Asn Phe Glu Asp 130 135 140 Tyr Val Asn Ala Leu Asn Ser TrpLys Lys Thr Pro Leu Ser Leu Arg 145 150 155 160 Ser Lys Arg Ser Gln AspArg Ile Arg Glu Leu Phe Ser Gln Ala Glu 165 170 175 Ser His Phe Arg AsnSer Met Pro Ser Phe Ala Val Ser Lys Phe Glu 180 185 190 Val Leu Phe LeuPro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu 195 200 205 Leu Leu LysAsp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser 210 215 220 Glu AspVal Ala Glu Phe Tyr Arg Arg Gln Leu Lys Leu Thr Gln Gln 225 230 235 240Tyr Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu Asn Gly Leu 245 250255 Arg Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg 260265 270 Glu Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr275 280 285 Asp Ile Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr ArgAsp 290 295 300 Ile Phe Thr Asp Pro Ile Phe Leu Leu Thr Thr Leu Gln LysTyr Gly 305 310 315 320 Pro Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg LysPro His Leu Phe 325 330 335 Asp Tyr Leu Gln Gly Ile Glu Phe His Thr ArgLeu Gln Pro Gly Tyr 340 345 350 Phe Gly Lys Asp Ser Phe Asn Tyr Trp SerGly Asn Tyr Val Glu Thr 355 360 365 Arg Pro Ser Ile Gly Ser Ser Lys ThrIle Thr Ser Pro Phe Tyr Gly 370 375 380 Asp Lys Ser Thr Glu Pro Val GlnLys Leu Ser Phe Asp Gly Gln Lys 385 390 395 400 Val Tyr Arg Thr Ile AlaAsn Thr Asp Val Ala Ala Trp Pro Asn Gly 405 410 415 Lys Val Tyr Leu GlyVal Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp 420 425 430 Gln Lys Asn GluThr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn 435 440 445 Gly His ValSer Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr 450 455 460 Thr AspGlu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala 465 470 475 480Glu Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr 485 490495 Trp Thr His Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys 500505 510 Ile Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala515 520 525 Ser Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu PheLeu 530 535 540 Lys Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr LeuAsn Ser 545 550 555 560 Ala Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile ArgTyr Ala Ser Thr 565 570 575 Thr Asn Leu Arg Leu Phe Val Gln Asn Ser AsnAsn Asp Phe Leu Val 580 585 590 Ile Tyr Ile Asn Lys Thr Met Asn Lys AspAsp Asp Leu Thr Tyr Gln 595 600 605 Thr Phe Asp Leu Ala Thr Thr Asn SerAsn Met Gly Phe Ser Gly Asp 610 615 620 Lys Asn Glu Leu Ile Ile Gly AlaGlu Ser Phe Val Ser Asn Glu Lys 625 630 635 640 Ile Tyr Ile Asp Lys IleGlu Phe Ile Pro Val Gln Leu 645 650 38 3044 DNA Artificial SequenceDescription of Artificial Sequence expression cassette 38 gcggccgcgttaacaagctt ctgacgtaag ggatgacgca cctgacgtaa gggatgacgc 60 acctgacgtaagggatgacg cacctgacgt aagggatgac gcactcgaga tccccatctc 120 cactgacgtaagggatgacg cacaatccca ctatccttcg caagaccctt cctctatata 180 aggaagttcatttcatttgg agaggacacg ctgacaagct agcttggctg caggtagatc 240 ctagaaccatcttccacaca ctcaagccac actattggag aacacacagg gacaacacac 300 cataagatccaagggaggcc tccgccgccg ccggtaacca ccccgcccct ctcctctttc 360 tttctccgtttttttttccg tctcggtctc gatctttggc cttggtagtt tgggtgggcg 420 agaggcggcttcgtgcgcgc ccagatcggt gcgcgggagg ggcgggatct cgcggctggg 480 gctctcgccggcgtggatcc ggcccggatc tcgcggggaa tggggctctc ggatgtagat 540 ctgcgatccgccgttgttgg gggagatgat ggggggttta aaatttccgc cgtgctaaac 600 aagatcaggaagaggggaaa agggcactat ggtttatatt tttatatatt tctgctgctt 660 cgtcaggcttagatgtgcta gatctttctt tcttcttttt gtgggtagaa tttgaatccc 720 tcagcattgttcatcggtag tttttctttt catgatttgt gacaaatgca gcctcgtgcg 780 gagcttttttgtaggtagaa gtgatcaacc atg gca aac cct aac aat cgt tcc 834 Met Ala AsnPro Asn Asn Arg Ser 1 5 gaa cac gac acc atc aag gtt act cca aac tct gagttg caa act aat 882 Glu His Asp Thr Ile Lys Val Thr Pro Asn Ser Glu LeuGln Thr Asn 10 15 20 cac aac cag tac cca ttg gct gac aat cct aac agt actctt gag gaa 930 His Asn Gln Tyr Pro Leu Ala Asp Asn Pro Asn Ser Thr LeuGlu Glu 25 30 35 40 ctt aac tac aag gag ttt ctc cgg atg acc gaa gat agctcc act gag 978 Leu Asn Tyr Lys Glu Phe Leu Arg Met Thr Glu Asp Ser SerThr Glu 45 50 55 gtt ctc gat aac tct aca gtg aag gac gct gtt gga act ggcatt agc 1026 Val Leu Asp Asn Ser Thr Val Lys Asp Ala Val Gly Thr Gly IleSer 60 65 70 gtt gtg gga cag att ctt gga gtg gtt ggt gtt cca ttc gct ggagct 1074 Val Val Gly Gln Ile Leu Gly Val Val Gly Val Pro Phe Ala Gly Ala75 80 85 ttg acc agc ttc tac cag tcc ttt ctc aac acc atc tgg cct tca gat1122 Leu Thr Ser Phe Tyr Gln Ser Phe Leu Asn Thr Ile Trp Pro Ser Asp 9095 100 gct gat ccc tgg aag gct ttc atg gcc caa gtg gaa gtc ttg atc gat1170 Ala Asp Pro Trp Lys Ala Phe Met Ala Gln Val Glu Val Leu Ile Asp 105110 115 120 aag aag atc gaa gag tat gcc aag tct aaa gcc ttg gct gag ttgcaa 1218 Lys Lys Ile Glu Glu Tyr Ala Lys Ser Lys Ala Leu Ala Glu Leu Gln125 130 135 ggt ttg cag aac aac ttc gag gat tac gtc aac gca ctc aac agctgg 1266 Gly Leu Gln Asn Asn Phe Glu Asp Tyr Val Asn Ala Leu Asn Ser Trp140 145 150 aag aaa act ccc ttg agt ctc agg tct aag cgt tcc cag gac cgtatt 1314 Lys Lys Thr Pro Leu Ser Leu Arg Ser Lys Arg Ser Gln Asp Arg Ile155 160 165 cgt gaa ctt ttc agc caa gcc gaa tcc cac ttc aga aac tcc atgcct 1362 Arg Glu Leu Phe Ser Gln Ala Glu Ser His Phe Arg Asn Ser Met Pro170 175 180 agc ttt gcc gtt tct aag ttc gag gtg ctc ttc ttg cca aca tacgca 1410 Ser Phe Ala Val Ser Lys Phe Glu Val Leu Phe Leu Pro Thr Tyr Ala185 190 195 200 caa gct gcc aac act cat ctc ttg ctt ctc aaa gac gct caggtg ttt 1458 Gln Ala Ala Asn Thr His Leu Leu Leu Leu Lys Asp Ala Gln ValPhe 205 210 215 ggt gag gaa tgg ggt tac tcc agt gaa gat gtt gcc gag ttctac cgt 1506 Gly Glu Glu Trp Gly Tyr Ser Ser Glu Asp Val Ala Glu Phe TyrArg 220 225 230 agg cag ctc aag ttg act caa cag tac aca gac cac tgc gtcaac tgg 1554 Arg Gln Leu Lys Leu Thr Gln Gln Tyr Thr Asp His Cys Val AsnTrp 235 240 245 tac aac gtt ggg ctc aat ggt ctt aga gga tct acc tac gacgca tgg 1602 Tyr Asn Val Gly Leu Asn Gly Leu Arg Gly Ser Thr Tyr Asp AlaTrp 250 255 260 gtg aag ttc aac agg ttt cgt aga gag atg acc ttg act gtgctc gat 1650 Val Lys Phe Asn Arg Phe Arg Arg Glu Met Thr Leu Thr Val LeuAsp 265 270 275 280 ctt atc gtt ctc ttt cca ttc tac gac att cgt ctt tactcc aaa ggc 1698 Leu Ile Val Leu Phe Pro Phe Tyr Asp Ile Arg Leu Tyr SerLys Gly 285 290 295 gtt aag aca gag ctg acc aga gac atc ttc acc gat cccatc ttc cta 1746 Val Lys Thr Glu Leu Thr Arg Asp Ile Phe Thr Asp Pro IlePhe Leu 300 305 310 ctt acg acc ctg cag aaa tac ggt cca act ttt ctc tccatt gag aac 1794 Leu Thr Thr Leu Gln Lys Tyr Gly Pro Thr Phe Leu Ser IleGlu Asn 315 320 325 agc atc agg aag cct cac ctc ttc gac tat ctg caa ggcatt gag ttt 1842 Ser Ile Arg Lys Pro His Leu Phe Asp Tyr Leu Gln Gly IleGlu Phe 330 335 340 cac acc agg ttg caa cct ggt tac ttc ggt aag gat tccttc aac tac 1890 His Thr Arg Leu Gln Pro Gly Tyr Phe Gly Lys Asp Ser PheAsn Tyr 345 350 355 360 tgg agc gga aac tac gtt gaa acc aga cca tcc atcgga tct agc aag 1938 Trp Ser Gly Asn Tyr Val Glu Thr Arg Pro Ser Ile GlySer Ser Lys 365 370 375 acc atc act tct cca ttc tac ggt gac aag agc actgag cca gtg cag 1986 Thr Ile Thr Ser Pro Phe Tyr Gly Asp Lys Ser Thr GluPro Val Gln 380 385 390 aag ttg agc ttc gat ggg cag aag gtg tat aga accatc gcc aat acc 2034 Lys Leu Ser Phe Asp Gly Gln Lys Val Tyr Arg Thr IleAla Asn Thr 395 400 405 gat gtt gca gct tgg cct aat ggc aag gtc tac cttgga gtt act aaa 2082 Asp Val Ala Ala Trp Pro Asn Gly Lys Val Tyr Leu GlyVal Thr Lys 410 415 420 gtg gac ttc tcc caa tac gac gat cag aag aac gagaca tct act caa 2130 Val Asp Phe Ser Gln Tyr Asp Asp Gln Lys Asn Glu ThrSer Thr Gln 425 430 435 440 acc tac gat agt aag agg aac aat ggc cat gtttcc gca caa gac tcc 2178 Thr Tyr Asp Ser Lys Arg Asn Asn Gly His Val SerAla Gln Asp Ser 445 450 455 att gac caa ctt cca cct gaa acc act gat gaacca ttg gag aag gct 2226 Ile Asp Gln Leu Pro Pro Glu Thr Thr Asp Glu ProLeu Glu Lys Ala 460 465 470 tac agt cac caa ctt aac tac gcc gaa tgc tttctc atg caa gac agg 2274 Tyr Ser His Gln Leu Asn Tyr Ala Glu Cys Phe LeuMet Gln Asp Arg 475 480 485 cgt ggc acc att ccg ttc ttt aca tgg act cacagg tct gtc gac ttc 2322 Arg Gly Thr Ile Pro Phe Phe Thr Trp Thr His ArgSer Val Asp Phe 490 495 500 ttt aac act atc gac gct gag aag att acc caactt ccc gtg gtc aag 2370 Phe Asn Thr Ile Asp Ala Glu Lys Ile Thr Gln LeuPro Val Val Lys 505 510 515 520 gct tat gcc ttg tcc agc gga gct tcc atcatt gaa ggt cca ggc ttc 2418 Ala Tyr Ala Leu Ser Ser Gly Ala Ser Ile IleGlu Gly Pro Gly Phe 525 530 535 acc ggt ggc aac ttg ctc ttc ctt aag gagtcc agc aac tcc atc gcc 2466 Thr Gly Gly Asn Leu Leu Phe Leu Lys Glu SerSer Asn Ser Ile Ala 540 545 550 aag ttc aaa gtg aca ctt aac tca gca gccttg ctc caa cgt tac agg 2514 Lys Phe Lys Val Thr Leu Asn Ser Ala Ala LeuLeu Gln Arg Tyr Arg 555 560 565 gtt cgt atc aga tac gca agc act acc aatctt cgc ctc ttt gtc cag 2562 Val Arg Ile Arg Tyr Ala Ser Thr Thr Asn LeuArg Leu Phe Val Gln 570 575 580 aac agc aac aat gat ttc ctt gtc atc tacatc aac aag act atg aac 2610 Asn Ser Asn Asn Asp Phe Leu Val Ile Tyr IleAsn Lys Thr Met Asn 585 590 595 600 aaa gac gat gac ctc acc tac caa acattc gat ctt gcc act acc aat 2658 Lys Asp Asp Asp Leu Thr Tyr Gln Thr PheAsp Leu Ala Thr Thr Asn 605 610 615 agt aac atg gga ttc tct ggt gac aagaac gag ctg atc ata ggt gct 2706 Ser Asn Met Gly Phe Ser Gly Asp Lys AsnGlu Leu Ile Ile Gly Ala 620 625 630 gag agc ttt gtc tct aat gag aag atttac ata gac aag atc gag ttc 2754 Glu Ser Phe Val Ser Asn Glu Lys Ile TyrIle Asp Lys Ile Glu Phe 635 640 645 att cca gtt caa ctc taatagatcccccgggctgc aggaattctg catgcgtttg 2809 Ile Pro Val Gln Leu 650 gacgtatgctcattcaggtt ggagccaatt tggttgatgt gtgtgcgagt tcttgcgagt 2869 ctgatgagacatctctgtat tgtgtttctt tccccagtgt tttctgtact tgtgtaatcg 2929 gctaatcgccaacagattcg gcgatgaata aatgagaaat aaattgttct gattttgagt 2989 gcaaaaaaaaaggaattaga tctgtgtgtg ttttttggat ccccggggcg gccgc 3044 39 653 PRTArtificial Sequence PRT (1)..(653) variant Cry3Bb1 coding sequenceencoding v11231 39 Met Ala Asn Pro Asn Asn Arg Ser Glu His Asp Thr IleLys Val Thr 1 5 10 15 Pro Asn Ser Glu Leu Gln Thr Asn His Asn Gln TyrPro Leu Ala Asp 20 25 30 Asn Pro Asn Ser Thr Leu Glu Glu Leu Asn Tyr LysGlu Phe Leu Arg 35 40 45 Met Thr Glu Asp Ser Ser Thr Glu Val Leu Asp AsnSer Thr Val Lys 50 55 60 Asp Ala Val Gly Thr Gly Ile Ser Val Val Gly GlnIle Leu Gly Val 65 70 75 80 Val Gly Val Pro Phe Ala Gly Ala Leu Thr SerPhe Tyr Gln Ser Phe 85 90 95 Leu Asn Thr Ile Trp Pro Ser Asp Ala Asp ProTrp Lys Ala Phe Met 100 105 110 Ala Gln Val Glu Val Leu Ile Asp Lys LysIle Glu Glu Tyr Ala Lys 115 120 125 Ser Lys Ala Leu Ala Glu Leu Gln GlyLeu Gln Asn Asn Phe Glu Asp 130 135 140 Tyr Val Asn Ala Leu Asn Ser TrpLys Lys Thr Pro Leu Ser Leu Arg 145 150 155 160 Ser Lys Arg Ser Gln AspArg Ile Arg Glu Leu Phe Ser Gln Ala Glu 165 170 175 Ser His Phe Arg AsnSer Met Pro Ser Phe Ala Val Ser Lys Phe Glu 180 185 190 Val Leu Phe LeuPro Thr Tyr Ala Gln Ala Ala Asn Thr His Leu Leu 195 200 205 Leu Leu LysAsp Ala Gln Val Phe Gly Glu Glu Trp Gly Tyr Ser Ser 210 215 220 Glu AspVal Ala Glu Phe Tyr Arg Arg Gln Leu Lys Leu Thr Gln Gln 225 230 235 240Tyr Thr Asp His Cys Val Asn Trp Tyr Asn Val Gly Leu Asn Gly Leu 245 250255 Arg Gly Ser Thr Tyr Asp Ala Trp Val Lys Phe Asn Arg Phe Arg Arg 260265 270 Glu Met Thr Leu Thr Val Leu Asp Leu Ile Val Leu Phe Pro Phe Tyr275 280 285 Asp Ile Arg Leu Tyr Ser Lys Gly Val Lys Thr Glu Leu Thr ArgAsp 290 295 300 Ile Phe Thr Asp Pro Ile Phe Leu Leu Thr Thr Leu Gln LysTyr Gly 305 310 315 320 Pro Thr Phe Leu Ser Ile Glu Asn Ser Ile Arg LysPro His Leu Phe 325 330 335 Asp Tyr Leu Gln Gly Ile Glu Phe His Thr ArgLeu Gln Pro Gly Tyr 340 345 350 Phe Gly Lys Asp Ser Phe Asn Tyr Trp SerGly Asn Tyr Val Glu Thr 355 360 365 Arg Pro Ser Ile Gly Ser Ser Lys ThrIle Thr Ser Pro Phe Tyr Gly 370 375 380 Asp Lys Ser Thr Glu Pro Val GlnLys Leu Ser Phe Asp Gly Gln Lys 385 390 395 400 Val Tyr Arg Thr Ile AlaAsn Thr Asp Val Ala Ala Trp Pro Asn Gly 405 410 415 Lys Val Tyr Leu GlyVal Thr Lys Val Asp Phe Ser Gln Tyr Asp Asp 420 425 430 Gln Lys Asn GluThr Ser Thr Gln Thr Tyr Asp Ser Lys Arg Asn Asn 435 440 445 Gly His ValSer Ala Gln Asp Ser Ile Asp Gln Leu Pro Pro Glu Thr 450 455 460 Thr AspGlu Pro Leu Glu Lys Ala Tyr Ser His Gln Leu Asn Tyr Ala 465 470 475 480Glu Cys Phe Leu Met Gln Asp Arg Arg Gly Thr Ile Pro Phe Phe Thr 485 490495 Trp Thr His Arg Ser Val Asp Phe Phe Asn Thr Ile Asp Ala Glu Lys 500505 510 Ile Thr Gln Leu Pro Val Val Lys Ala Tyr Ala Leu Ser Ser Gly Ala515 520 525 Ser Ile Ile Glu Gly Pro Gly Phe Thr Gly Gly Asn Leu Leu PheLeu 530 535 540 Lys Glu Ser Ser Asn Ser Ile Ala Lys Phe Lys Val Thr LeuAsn Ser 545 550 555 560 Ala Ala Leu Leu Gln Arg Tyr Arg Val Arg Ile ArgTyr Ala Ser Thr 565 570 575 Thr Asn Leu Arg Leu Phe Val Gln Asn Ser AsnAsn Asp Phe Leu Val 580 585 590 Ile Tyr Ile Asn Lys Thr Met Asn Lys AspAsp Asp Leu Thr Tyr Gln 595 600 605 Thr Phe Asp Leu Ala Thr Thr Asn SerAsn Met Gly Phe Ser Gly Asp 610 615 620 Lys Asn Glu Leu Ile Ile Gly AlaGlu Ser Phe Val Ser Asn Glu Lys 625 630 635 640 Ile Tyr Ile Asp Lys IleGlu Phe Ile Pro Val Gln Leu 645 650 40 32 DNA Artificial SequenceDescription of Artificial Sequence synthetic oligonucleotide 40taggcctcca tccatggcaa accctaacaa tc 32 41 42 DNA Artificial SequenceDescription of Artificial Sequence synthetic oligonucleotide 41tcccatcttc ctacttagca ccctgcagaa atacggtcca ac 42 42 28 DNA ArtificialSequence Description of Artificial Sequence synthetic oligonucleotide 42gacctcacct accaaacatt cgatcttg 28 43 25 DNA Artificial SequenceDescription of Artificial Sequence synthetic oligonucleotide 43cgagttctac cgtaggcagc tcaag 25

What is claimed is:
 1. A modified polynucleotide comprising a nucleotidesequence as set forth in SEQ ID NO:9.
 2. A cell comprising the modifiedpolynucleotide of claim
 1. 3. The cell according to claim 2 wherein saidcell is a plant cell or a bacterial cell.
 4. The cell according to claim3 wherein said plant cell is a maize cell.
 5. A method of producing atransformed cell, said method comprising introducing a modifiedpolynucleotide into a cell, wherein said modified polynucleotidecomprises a nucleotide sequence as set forth in SEQ ID NO:9, and whereinsaid cell is a plant cell or a microbial cell.
 6. The method of claim 5wherein said cell is a plant cell.
 7. The method of claim 5 wherein saidplant cell is a maize cell.
 8. A method of producing a transformed maizeplant comprising introducing the nucleotide sequence as set forth in SEQID NO:9 into a maize plant cell, selecting a transformed maize plantcell, and regenerating a maize plant from said transformed maize plantcell, wherein said maize plant comprises said nucleotide sequence.
 9. Aplant comprising the nucleotide sequence as set forth in SEQ ID NO:9.10. A seed or progeny from the plant of claim 9, wherein said seed orprogeny comprises said nucleotide sequence.
 11. A plant grown from theseed of claim
 10. 12. A method of controlling Coleopteran insectinfestation in a field of crop plants comprising providing to saidColeopteran insect a transgenic plant on which said Coleopteran insectfeeds, said transgenic plant comprising a polynucleotide sequenceencoding the peptide as set forth in SEQ ID NO:10.
 13. The method ofclaim 12, wherein said polynucleotide sequence comprises the nucleotidesequence as set forth in SEQ ID NO:9.
 14. A vector comprising anucleotide sequence encoding the peptide as set forth in SEQ ID NO:10,wherein said nucleotide sequence is contained within an expressioncassette, said expression cassette comprising from about nucleotideposition 14 to about nucleotide position 3020 as set forth in SEQ IDNO:19.
 15. The method of claim 14, wherein said nucleotide sequenceencoding the peptide comprises the sequence as set forth in SEQ ID NO:9.